Cripto tumour polypeptide

ABSTRACT

Compositions and methods for the therapy and diagnosis of cancer, particularly lung, colon, colorectal and breast cancer, are disclosed. Illustrative compositions comprise one or more Cripto tumor polypeptides, immunogenic portions thereof, polynucleotides that encode such polypeptides, antigen presenting cell that expresses such polypeptides, and T cells that are specific for cells expressing such polypeptides. The disclosed compositions are useful, for example, in the diagnosis, prevention and/or treatment of diseases, particularly lung, colon, colorectal and breast cancer.

This is the national phase under 35 U.S.C. § 371 of PCT InternationalApplication PCT/EP01/09646, filed 20 Aug. 2001, which claims benefitfrom Great Britain Application No.: GB 0020953.6, filed 24 Aug. 2000.

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to therapy of cancer, such ascolon, colorectal, breast, bladder, lung and endometrial cancer. Theinvention is more specifically related to polypeptides, comprising atleast a portion of a cripto protein tumor protein, and topolynucleotides encoding such polypeptides, in particular pharmaceuticalcompositions, e.g., vaccines, and other compositions for the treatmentof cancer that are cripto—expressing carcinomas such as certainnon-small long cell carcinoma, breast, colon, colorectal cancer.

BACKGROUND OF THE INVENTION

CRIPTO is a 188 aa protein shares homologies with the epidermal growthfactor (EGF) family (EMBO Journal (1989) Vol 8 (7) pp1987-1991).

huCRIPTO mRNA is detected only in undifferentiated cells and disappearafter cell differentiation mCRIPTO is expressed during pregnancy andlactation (induces branching morphogenesis in mammary epithelial cells)and is suspected to be an autocrine growth factor for normal breastcells. CRIPTO is required for correct orientation of theanterior-posterior axis in the mouse embryo.

Human cripto gene has been expressed U.S. Pat. No. 5,654,140.

Cancer is a significant health problem throughout the world. Althoughadvances have been made in detection and therapy of cancer, no vaccineor other universally successful method for prevention and/or treatmentis currently available. Current therapies, which are generally based ona combination of chemotherapy or surgery and radiation, continue toprove inadequate in many patients.

Colon cancer is the second most frequently diagnosed malignancy in theUnited Sates as well as the second most common cause of cancer death. Anestimated 95,600 new cases of colon cancer will have been diagnosed in1998, with an estimated 47,700 deaths. The five-year survival rate forpatients with colorectal cancer detected in an early-localised stage is92%, unfortunately, only 37% of colorectal cancer is diagnosed at thisstage. The survival stage. The survival rate drops to 64% if the canceris allowed to spread to adjacent organs or lymph nodes, and to 7% inpatients with distant metastases.

The prognosis of colon cancer is directly related to the degree ofpenetration of the tumour through the bowel wall, to the level ofmetastasis, to the presence or absence of nodal involvement,consequently, early detection and treatment are especially important.Currently, diagnosis is aided by the use of screening assays for fecaloccult blood, sigmoidoscopy, colonoscopy and double contrast bariumenemas. Treatment regimens are determined by the type and stage of thecancer, and include surgery, radiation therapy and/or chemotherapy.Recurrence following surgery (the most common form of therapy) is amajor problem and is often the ultimate cause of death. In spite ofconsiderable research into therapies for the disease, colon cancerremains difficult to diagnose and treat. Accordingly, there is a need inthe art for improved methods for treating such cancers. The presentinvention fulfils these needs and further provides other relatedadvantages.

Breast cancer is a significant health problem for women in the UnitedStates and throughout the world. Although advances have been made indetection and treatment of the disease, breast cancer remains the secondleading cause of cancer-related deaths in women, affecting more than180,000 women in the United States each year. For women in NorthAmerica, the lifetime odds of getting breast cancer are now one ineight.

No vaccine or other universally successful method for the prevention ortreatment of breast cancer is currently available. Management of thedisease currently relies on a combination of early diagnosis (throughroutine breast screening procedures) and aggressive treatment which mayincluding one or more of a variety of treatments such as surgery,radiotherapy, chemotherapy and hormone therapy. The course of treatmentfor a particular breast cancer is often selected based on a variety ofprognostic parameters, including an analysis of specific tumour markers.See, eg Porter-Jordan and Lippman, Breast Cancer 8:73-100 (1994).However, the use of established markers often leads to a result thatthis is difficult to interpret, and the high mortality observed inbreast cancer patients indicates that improvements are needed in thetreatment and prevention of the disease.

Lung cancer is the primary cause of cancer death among both men andwomen in the US, with an estimated 172,000 new cases being reported in1994. The five-year survival rate among all lung cancer patients,regardless of the state of disease at diagnosis is only 13%. Thiscontrasts with a five-year survival rate of 46% among cases detectedwhile the disease is still localised. However, only 16% of lung cancersare discovered before the disease has spread.

In spite of considerable research into therapies for these and othercancers, breast, colon and colorectal remains difficult to diagnose andtreat effectively. Accordingly, there is a need in the art for improvedmethods for treating and preventing such cancers. The present inventionfulfills these needs and further provides other related advantages.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides polynucleotidecompositions comprising a sequence selected from the group consistingof:

-   -   (a) sequences consisting of at least 20 contiguous residues of a        sequence provided in SEQ ID NO: 1 or 3 not being the full        sequence of ID NO:1 or 3.    -   (b) sequences consisting of Sequence ID NO: 1 or 3 or sequences        that consist of at least 20 contiguos nucleotides provided in        Seq ID No 1 or 3 and a polynucleotide that encodes for a        heterologous fusion partner.

The present invention, in another aspect, provides polypeptidecompositions comprising an amino acid sequence that is encoded by apolynucleotide sequence described above.

The polypeptides and/or polynucleotides of the present invention areimmunogenic, i.e., they are capable of eliciting an immune response,particularly a humoral and/or cellular immune response, when ifnecessary, they are conjugated to a suitable carrier and/or adjuvanted.

The present invention further provides fragments, variants and/orderivatives of the disclosed polypeptide and/or polynucleotidesequences, wherein the fragments, variants and/or derivatives preferablyhave a level of immunogenic activity of at least about 50%, preferablyat least about 70% and more preferably at least about 90% of the levelof immunogenic activity of a polypeptide sequence set forth in SEQ IDNo: 2 or 4 or a polypeptide sequence encoded by a polynucleotidesequence set forth in SEQ ID NO: 1 or 3.

The present invention further provides polynucleotides that encode apolypeptide described above, expression vectors comprising suchpolynucleotides and host cells transformed or transfected with suchexpression vectors.

Within other aspects, the present invention provides pharmaceuticalcompositions comprising a polypeptide or polynucleotide as describedabove and a physiologically acceptable carrier.

Within a related aspect of the present invention, the pharmaceuticalcompositions, e.g., vaccine compositions, are provided for prophylacticor therapeutic applications. Such compositions generally comprise animmunogenic polypeptide or polynucleotide of the invention and animmunostimulant, such as an adjuvant.

Within further aspects, the present invention provides pharmaceuticalcompositions comprising: (a) an antigen presenting cell that expresses apolypeptide as described above and (b) a pharmaceutically acceptablecarrier or excipient. Illustrative antigen presenting cells includedendritic cells, macrophages, monocytes, fibroblasts and B cells.

Within related aspects, pharmaceutical compositions are provided thatcomprise: (a) an antigen presenting cell that expresses a polypeptide asdescribed above and (b) an immunostimulant.

The present invention further provides, in other aspects, fusionproteins that comprise at least one polypeptide as described above, aswell as polynucleotides encoding such fusion proteins, typically in theform of pharmaceutical compositions, e.g., vaccine compositions,comprising a physiologically acceptable carrier and/or animmunostimulant. The fusion proteins may comprise multiple immunogenicpolypeptides or portions/variants thereof, as described herein, and mayfurther comprise one or more polypeptide segments for facilitating theexpression, purification and/or immunogenicity of the polypeptide(s).

Within further aspects, the present invention provides methods forstimulating an immune response in a patient, preferably a T cellresponse in a human patient, comprising administering a pharmaceuticalcomposition described herein. The patient may be afflicted with lung orcolon cancer or colorectal cancer or breast cancer, in which case themethods provide treatment for the disease, or patient considered at riskfor such a disease may be treated prophylactically. In particular thepatient will be afflicted with a tumour expressing cripto antigens.

Within further aspects, the present invention provides methods forinhibiting the development of a cancer in a patient, comprisingadministering to a patient a pharmaceutical composition as recitedabove. The patient may be afflicted with lung, colon, colorectal orbreast cancer, in which case the methods provide treatment for thedisease, or patient considered at risk for such a disease may be treatedprophylactically.

The present invention further provides, within other aspects, methodsfor removing tumor cells from a biological sample, comprising contactinga biological sample with T cells that specifically react with apolypeptide of the present invention, wherein the step of contacting isperformed under conditions and for a time sufficient to permit theremoval of cells expressing the protein from the sample.

Within related aspects, methods are provided for inhibiting thedevelopment of a cancer in a patient, comprising administering to apatient a biological sample treated as described above.

Methods are further provided, within other aspects, for stimulatingand/or expanding T cells specific for a polypeptide of the presentinvention, comprising contacting T cells with one or more of: (i) apolypeptide as described above; (ii) a polynucleotide encoding such apolypeptide; and/or (iii) an antigen presenting cell that expresses sucha polypeptide; under conditions and for a time sufficient to permit thestimulation and/or expansion of T cells. Isolated T cell populationscomprising T cells prepared as described above are also provided.

Within further aspects, the present invention provides methods forinhibiting the development of a cancer in a patient, comprisingadministering to a patient an effective amount of a T cell population asdescribed above.

The present invention further provides methods for inhibiting thedevelopment of a cancer in a patient, comprising the steps of: (a)incubating CD4+ and/or CD8+ T cells isolated from a patient with one ormore of: (i) a polypeptide comprising at least an immunogenic portion ofa cripto polypeptide disclosed herein; (ii) a polynucleotide encodingsuch a polypeptide; and (iii) an antigen-presenting cell that expressedsuch a polypeptide; and (b) administering to the patient an effectiveamount of the proliferated T cells, and thereby inhibiting thedevelopment of a cancer in the patient. Proliferated cells may, but neednot, be cloned prior to administration to the patient.

These and other aspects of the present invention will become apparentupon reference to the following detailed description. All referencesdisclosed herein are hereby incorporated by reference in their entiretyas if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE IDENTIFIERS

SEQ ID NO: 1 Cripto 1 Polynucleotide

SEQ ID NO: 2 Cripto 3 Polynucleotide

SEQ ID NO: 2 Cripto 3 Polypeptide 1

SEQ ID NO: 3 Cripto 1 Polypeptide

SEQ ID NO: 4 Cripto 3 Polypeptide

SEQ ID NO: 5 Cripto Polynucleotide as described in US 5654 140

SEQ ID NO: 6 Cripto Polynucleotide as described in US 5654 140

SEQ ID NOS: 7-10 PCR primers

SEQ ID NOS: 11 & 12 synthetic Cripto 1 peptides

SEQ ID NOS: 13-94 Epitopes from Cripto 1 and 3

SEQ ID NO: 95 Cripto 1 variant Polynucleotide

SEQ ID NO: 96 Cripto 1 variant Polypeptide

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed generally to compositions and theiruse in the therapy and diagnosis of cancer, particularly criptoexpressing cancer and metastases including Cripto expressing lung,colon, colorectal and breast cancers. As described further below,illustrative compositions of the present invention include, but are notrestricted to, polypeptides, particularly immunogenic polypeptides,polynucleotides encoding such polypeptides, antibodies and other bindingagents, antigen presenting cells (APCs) and immune system cells (e.g., Tcells).

The practice of the present invention will employ, unless indicatedspecifically to the contrary, conventional methods of virology,immunology, microbiology, molecular biology and recombinant DNAtechniques within the skill of the art, many of which are describedbelow for the purpose of illustration. Such techniques are explainedfully in the literature. See, e.g., Sambrook, et al. Molecular Cloning:A Laboratory Manual (2nd Edition, 1989); Maniatis et al. MolecularCloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach,vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed.,1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985);Transcription and Translation (B. Hames & S. Higgins, eds., 1984);Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guideto Molecular Cloning (1984).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

Polypeptide Compositions

As used herein, the term “polypeptide” is used in its conventionalmeaning, i.e. as a sequence of amino acids. The polypeptides are notlimited to a specific length of the product; thus, peptides,oligopeptides, and proteins are included within the definition ofpolypeptide, and such terms may be used interchangeably herein unlessspecifically indicated otherwise. This term also does not refer to orexclude post-expression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like, as well asother modifications known in the art, both naturally occurring andnon-naturally occurring. A polypeptide may be an entire protein, or asubsequence thereof. Particular polypeptides of interest in the contextof this invention are amino acid subsequences comprising epitopes, i.e.antigenic determinants substantially responsible for the immunogenicproperties of a polypeptide and being capable of evoking an immuneresponse

Particularly illustrative polypeptides of the present invention comprisea sequence of at least 10 contiguous amino acids, preferably 20, morepreferably 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,160, 170, 180 amino acids of the cirpto protein of ID NO: 2 or 4.

In certain preferred embodiments, the polypeptides of the invention areimmunogenic, i.e., they react detectably within an immunoassay (such asan ELISA or T-cell stimulation assay) with antisera and/or T-cells froma patient with cripto expressing cancer. Screening for immunogenicactivity can be performed using techniques well known to the skilledartisan. For example, such screens can be performed using methods suchas those described in Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, 1988. In one illustrative example, apolypeptide may be immobilized on a solid support and contacted withpatient sera to allow binding of antibodies within the sera to theimmobilized polypeptide. Unbound sera may then be removed and boundantibodies detected using, for example, ¹²⁵I-labeled Protein A.

As would be recognized by the skilled artisan, immunogenic portions ofthe polypeptides disclosed herein are also encompassed by the presentinvention. An “immunogenic portion,” as used herein, is a fragment of animmunogenic polypeptide of the invention that itself is immunologicallyreactive (i.e., specifically binds) with the B-cells and/or T-cellsurface antigen receptors that recognize the polypeptide. Immunogenicportions may generally be identified using well known techniques, suchas those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247(Raven Press, 1993) and references cited therein. Such techniquesinclude screening polypeptides for the ability to react withantigen-specific antibodies, antisera and/or T-cell lines or clones. Asused herein, antisera and antibodies are “antigen-specific” if theyspecifically bind to an antigen (i.e., they react with the protein in anELISA or other immunoassay, and do not react detectably with unrelatedproteins). Such antisera and antibodies may be prepared as describedherein, and using well-known techniques.

In one preferred embodiment, an immunogenic portion of a polypeptide ofthe present invention is a portion that reacts with antisera and/orT-cells at a level that is not substantially less than the reactivity ofthe full-length polypeptide (e.g., in an ELISA and/or T-cell reactivityassay). Preferably, the level of immunogenic activity of the immunogenicportion is at least about 50%, preferably at least about 70% and mostpreferably greater than about 90% of the immunogenicity for thefull-length polypeptide. In some instances, preferred immunogenicportions will be identified that have a level of immunogenic activitygreater than that of the corresponding full-length polypeptide, e.g.,having greater than about 100% or 150% or more immunogenic activity.

In certain other embodiments, illustrative immunogenic portions mayinclude peptides in which an N-terminal leader sequence and/ortransmembrane domain have been deleted. Other illustrative immunogenicportions will contain a small N- and/or C-terminal deletion (e.g., 1-30amino acids, preferably 5-15 amino acids), relative to the matureprotein.

In another embodiment, a polypeptide composition of the invention mayalso comprise one or more polypeptides that are immunologically reactivewith T cells and/or antibodies generated against a polypeptide of theinvention, particularly a polypeptide having an amino acid sequencedisclosed herein, or to an immunogenic fragment or variant thereof.

In another embodiment of the invention, polypeptides are provided thatcomprise one or more polypeptides that are capable of eliciting T cellsand/or antibodies that are immunologically reactive with one or morepolypeptides described herein, or one or more polypeptides encoded bycontiguous nucleic acid sequences contained in the polynucleotidesequences disclosed herein, or immunogenic fragments or variantsthereof, or to one or more nucleic acid sequences which hybridize to oneor more of these sequences under conditions of moderate to highstringency.

The present invention, in another aspect, provides polypeptide fragmentscomprising at least about 5, 10, 15, 20, 25, 50, 100, or 150 contiguousamino acids, or more, including all intermediate lengths, of apolypeptide compositions set forth herein, such as those set forth inSEQ ID NO: 2 or 4 or those encoded by a polynucleotide sequence setforth in a sequence of SEQ ID NO: 1 or 3. It is preferred that thepolypeptides comprise at least one preferably a pluarality of epitopesas set forth in sequence ID no 13 to 94. Optionally the frgaments arefused or otherwise conjugated to a heterologous carrier.

In another aspect, the present invention provides variants of thepolypeptide compositions described herein. Polypeptide variantsgenerally encompassed by the present invention will typically exhibit atleast about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or more identity (determined as described below), along itslength, to a polypeptide sequences set forth herein.

In one preferred embodiment, the polypeptide fragments and variantsprovide by the present invention are immunologically reactive with anantibody and/or T-cell that reacts with a full-length polypeptidespecifically set for the herein.

In another preferred embodiment, the polypeptide fragments and variantsprovided by the present invention exhibit a level of immunogenicactivity of at least about 50%, preferably at least about 70%, and mostpreferably at least about 90% or more of that exhibited by a full-lengthpolypeptide sequence specifically set forth herein.

A polypeptide “variant,” as the term is used herein, is a polypeptidethat typically differs from a polypeptide specifically disclosed hereinin one or more substitutions, deletions, additions and/or insertions.Such variants may be naturally occurring or may be syntheticallygenerated, for example, by modifying one or more of the abovepolypeptide sequences of the invention and evaluating their immunogenicactivity as described herein and/or using any of a number of techniqueswell known in the art.

For example, certain illustrative variants of the polypeptides of theinvention include those in which one or more portions, such as anN-terminal leader sequence or transmembrane domain, have been removed.Other illustrative variants include variants in which a small portion(e.g., 1-30 amino acids, preferably 5-15 amino acids) has been removedfrom the N- and/or C-terminal of the mature protein.

In many instances, a variant will contain conservative substitutions. A“conservative substitution” is one in which an amino acid is substitutedfor another amino acid that has similar properties, such that oneskilled in the art of peptide chemistry would expect the secondarystructure and hydropathic nature of the polypeptide to be substantiallyunchanged. As described above, modifications may be made in thestructure of the polynucleotides and polypeptides of the presentinvention and still obtain a functional molecule that encodes a variantor derivative polypeptide with desirable characteristics, e.g., withimmunogenic characteristics. When it is desired to alter the amino acidsequence of a polypeptide to create an equivalent, or even an improved,immunogenic variant or portion of a polypeptide of the invention, oneskilled in the art will typically change one or more of the codons ofthe encoding DNA sequence according to Table 1.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated that various changes may bemade in the peptide sequences of the disclosed compositions, orcorresponding DNA sequences which encode said peptides withoutappreciable loss of their biological utility or activity.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GGU Cysteine Cys CUGG UGU Aspartic acid Asp D GAG GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUG AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporated herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like. Each amino acid has been assigned a hydropathicindex on the basis of its hydrophobicity and charge characteristics(Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e. still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those within ±1 are particularly preferred, and thosewithin ±0.5 are even more particularly preferred. It is also understoodin the art that the substitution of like amino acids can be madeeffectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101(specifically incorporated herein by reference in its entirety), statesthat the greatest local average hydrophilicity of a protein, as governedby the hydrophilicity of its adjacent amino acids, correlates with abiological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions that take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

In addition, any polynucleotide may be further modified to increasestability in vivo. Possible modifications include, but are not limitedto, the addition of flanking sequences at the 5′ and/or 3′ ends; the useof phosphorothioate or 2′ O-methyl rather than phosphodiesteraselinkages in the backbone; and/or the inclusion of nontraditional basessuch as inosine, queosine and wybutosine, as well as acetyl-methyl-,thio- and other modified forms of adenine, cytidine, guanine, thymineand uridine.

Amino acid substitutions may further be made on the basis of similarityin polarity, charge, solubility, hydrophobicity, hydrophilicity and/orthe amphipathic nature of the residues. For example, negatively chargedamino acids include aspartic acid and glutamic acid; positively chargedamino acids include lysine and arginine; and amino acids with unchargedpolar head groups having similar hydrophilicity values include leucine,isoleucine and valine; glycine and alanine; asparagine and glutamine;and serine, threonine, phenylalanine and tyrosine. Other groups of aminoacids that may represent conservative changes include: (1) ala, pro,gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile,leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Avariant may also, or alternatively, contain nonconservative changes. Ina preferred embodiment, variant polypeptides differ from a nativesequence by substitution, deletion or addition of five amino acids orfewer. Variants may also (or alternatively) be modified by, for example,the deletion or addition of amino acids that have minimal influence onthe immunogenicity, secondary structure and hydropathic nature of thepolypeptide.

As noted above, polypeptides may comprise a signal (or leader) sequenceat the N-terminal end of the protein, which co-translationally orpost-translationally directs transfer of the protein. The polypeptidemay also be conjugated to a linker or other sequence for ease ofsynthesis, purification or identification of the polypeptide (e.g.,poly-His), or to enhance binding of the polypeptide to a solid support.For example, a polypeptide may be conjugated to an immunoglobulin Fcregion.

When comparing polypeptide sequences, two sequences are said to be“identical” if the sequence of amino acids in the two sequences is thesame when aligned for maximum correspondence, as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W.and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA80:726-730.

Alternatively, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman (1981)Add. APL. Math 2:482, by the identity alignment algorithm of Needlemanand Wunsch (1970) J. Mol. Biol. 48:443, by the search for similaritymethods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT,BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or byinspection.

One preferred example of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al. (1977)Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol.215:403-410, respectively. BLAST and BLAST 2.0 can be used, for examplewith the parameters described herein, to determine percent sequenceidentity for the polynucleotides and polypeptides of the invention.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. For amino acid sequences,a scoring matrix can be used to calculate the cumulative score.Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment.

In one preferred approach, the “percentage of sequence identity” isdetermined by comparing two optimally aligned sequences over a window ofcomparison of at least 20 positions, wherein the portion of thepolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the referencesequence (i.e., the window size) and multiplying the results by 100 toyield the percentage of sequence identity.

Within other illustrative embodiments, a polypeptide may be a fusionpolypeptide that comprises multiple polypeptides as described herein, orthat comprises at least one polypeptide as described herein and anunrelated sequence, such as a known tumor protein. A fusion partner may,for example, assist in providing T helper epitopes (an immunologicalfusion partner), preferably T helper epitopes recognized by humans, ormay assist in expressing the protein (an expression enhancer) at higheryields than the native recombinant protein. Certain preferred fusionpartners are both immunological and expression enhancing fusionpartners. Other fusion partners may be selected so as to increase thesolubility of the polypeptide or to enable the polypeptide to betargeted to desired intracellular compartments. Still further fusionpartners include affinity tags, which facilitate purification of thepolypeptide.

Fusion polypeptides may generally be prepared using standard techniques,including chemical conjugation. Preferably, a fusion polypeptide isexpressed as a recombinant polypeptide, allowing the production ofincreased levels, relative to a non-fused polypeptide, in an expressionsystem. Briefly, DNA sequences encoding the polypeptide components maybe assembled separately, and ligated into an appropriate expressionvector. The 3′ end of the DNA sequence encoding one polypeptidecomponent is ligated, with or without a peptide linker, to the 5′ end ofa DNA sequence encoding the second polypeptide component so that thereading frames of the sequences are in phase. This permits translationinto a single fusion polypeptide that retains the biological activity ofboth component polypeptides.

A peptide linker sequence may be employed to separate the first andsecond polypeptide components by a distance sufficient to ensure thateach polypeptide folds into its secondary and tertiary structures. Sucha peptide linker sequence is incorporated into the fusion polypeptideusing standard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180.The linker sequence may generally be from 1 to about 50 amino acids inlength. Linker sequences are not required when the first and secondpolypeptides have non-essential N-terminal amino acid regions that canbe used to separate the functional domains and prevent stericinterference.

The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located only 5′ to theDNA sequence encoding the first polypeptides. Similarly, stop codonsrequired to end translation and transcription termination signals areonly present 3′ to the DNA sequence encoding the second polypeptide.

The fusion polypeptide can comprise a polypeptide as described hereintogether with an unrelated immunogenic protein, such as an immunogenicprotein capable of eliciting a recall response. Examples of suchproteins include tetanus, tuberculosis and hepatitis proteins (see, forexample, Stoute et al. New Engl. J. Med., 336:86-91, 1997).

In one preferred embodiment, the immunological fusion partner is derivedfrom a Mycobacterium sp., such as a Mycobacterium tuberculosis-derivedRa12 fragment. Ra12 compositions and methods for their use in enhancingthe expression and/or immunogenicity of heterologouspolynucleotide/polypeptide sequences is described in U.S. PatentApplication No. 60/158,585, the disclosure of which is incorporatedherein by reference in its entirety. Briefly, Ra12 refers to apolynucleotide region that is a subsequence of a Mycobacteriumtuberculosis MTB32A nucleic acid. MTB32A is a serine protease of 32 KDmolecular weight encoded by a gene in virulent and avirulent strains ofM. tuberculosis. The nucleotide sequence and amino acid sequence ofMTB32A have been described (for example, U.S. Patent Application No.60/158,585; see also, Skeiky et al., Infection and Immun. (1999)67:3998-4007, incorporated herein by reference). C-terminal fragments ofthe MTB32A coding sequence express at high levels and remain as asoluble polypeptides throughout the purification process. Moreover, Ra12may enhance the immunogenicity of heterologous immunogenic polypeptideswith which it is fused. One preferred Ra12 fusion polypeptide comprisesa 14 KD C-terminal fragment corresponding to amino acid residues 192 to323 of MTB32A. Other preferred Ra12 polynucleotides generally compriseat least about 15 consecutive nucleotides, at least about 30nucleotides, at least about 60 nucleotides, at least about 100nucleotides, at least about 200 nucleotides, or at least about 300nucleotides that encode a portion of a Ra12 polypeptide. Ra12polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes a Ra12 polypeptide or a portion thereof) or maycomprise a variant of such a sequence. Ra12 polynucleotide variants maycontain one or more substitutions, additions, deletions and/orinsertions such that the biological activity of the encoded fusionpolypeptide is not substantially diminished, relative to a fusionpolypeptide comprising a native Ra12 polypeptide. Variants preferablyexhibit at least about 70% identity, more preferably at least about 80%identity and most preferably at least about 90% identity to apolynucleotide sequence that encodes a native Ra12 polypeptide or aportion thereof.

Within other preferred embodiments, an immunological fusion partner isderived from protein D, a surface protein of the gram-negative bacteriumHaemophilus influenza B (WO 91/18926). Preferably, a protein Dderivative comprises approximately the first third of the protein (e.g.,the first N-terminal 100-110 amino acids), and a protein D derivativemay be lipidated. Within certain preferred embodiments, the first 109residues of a Lipoprotein D fusion partner is included on the N-terminusto provide the polypeptide with additional exogenous T-cell epitopes andto increase the expression level in E. coli (thus functioning as anexpression enhancer). The lipid tail ensures optimal presentation of theantigen to antigen presenting cells. Other fusion partners include thenon-structural protein from influenzae virus, NS1 (hemaglutinin).Typically, the N-terminal 81 amino acids are used, although differentfragments that include T-helper epitopes may be used.

In another embodiment, the immunological fusion partner is the proteinknown as LYTA, or a portion thereof (preferably a C-terminal portion).LYTA is derived from Streptococcus pneumoniae, which synthesizes anN-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytAgene; Gene 43:265-292, 1986). LYTA is an autolysin that specificallydegrades certain bonds in the peptidoglycan backbone. The C-terminaldomain of the LYTA protein is responsible for the affinity to thecholine or to some choline analogues such as DEAE. This property hasbeen exploited for the development of E. coli C-LYTA expressing plasmidsuseful for expression of fusion proteins. Purification of hybridproteins containing the C-LYTA fragment at the amino terminus has beendescribed (see Biotechnology 10:795-798, 1992). Within a preferredembodiment, a repeat portion of LYTA may be incorporated into a fusionpolypeptide. A repeat portion is found in the C-terminal region startingat residue 178. A particularly preferred repeat portion incorporatesresidues 188-305.

Yet another illustrative embodiment involves fusion polypeptides, andthe polynucleotides encoding them, wherein the fusion partner comprisesa targeting signal capable of directing a polypeptide to theendosomal/lysosomal compartment, as described in U.S. Pat. No.5,633,234. An immunogenic polypeptide of the invention, when fused withthis targeting signal, will associate more efficiently with MHC class IImolecules and thereby provide enhanced in vivo stimulation of CD4+T-cells specific for the polypeptide.

The cripto part of the fusion molecule may prefreably be the wholelength 188 aa protein of Cripto 1 or Cripto 3 or a fragment thereof asdescribed herein.

Polypeptides of the invention are prepared using any of a variety ofwell known synthetic and/or recombinant techniques, the latter of whichare further described below. Polypeptides, portions and other variantsgenerally less than about 150 amino acids can be generated by syntheticmeans, using techniques well known to those of ordinary skill in theart. In one illustrative example, such polypeptides are synthesizedusing any of the commercially available solid-phase techniques, such asthe Merrifield solid-phase synthesis method, where amino acids aresequentially added to a growing amino acid chain. See Merrifield, J. Am.Chem. Soc. 85:2149-2146, 1963. Equipment for automated synthesis ofpolypeptides is commercially available from suppliers such as PerkinElmer/Applied BioSystems Division (Foster City, Calif.), and may beoperated according to the manufacturer's instructions.

In general, polypeptide compositions (including fusion polypeptides) ofthe invention are isolated. An “isolated” polypeptide is one that isremoved from its original environment. For example, anaturally-occurring protein or polypeptide is isolated if it isseparated from some or all of the coexisting materials in the naturalsystem. Preferably, such polypeptides are also purified, e.g., are atleast about 90% pure, more preferably at least about 95% pure and mostpreferably at least about 99% pure.

Polynucleotide Compositions

The present invention, in other aspects, provides polynucleotidecompositions that encode for the polypeptides of the invention. Theterms “DNA” and “polynucleotide” are used essentially interchangeablyherein to refer to a DNA molecule that has been isolated free of totalgenomic DNA of a particular species. “Isolated,” as used herein, meansthat a polynucleotide is substantially away from other coding sequences,and that the DNA molecule does not contain large portions of unrelatedcoding DNA, such as large chromosomal fragments or other functionalgenes or polypeptide coding regions. Of course, this refers to the DNAmolecule as originally isolated, and does not exclude genes or codingregions later added to the segment by the hand of man.

As will be understood by those skilled in the art, the polynucleotidecompositions of this invention can include genomic sequences,extra-genomic and plasmid-encoded sequences and smaller engineered genesegments that express, or may be adapted to express, proteins,polypeptides, peptides and the like. Such segments may be naturallyisolated, or modified synthetically by the hand of man.

As will be also recognized by the skilled artisan, polynucleotides ofthe invention may be single-stranded (coding or antisense) ordouble-stranded, and may be DNA (genomic, cDNA or synthetic) or RNAmolecules. RNA molecules may include HnRNA molecules, which containintrons and correspond to a DNA molecule in a one-to-one manner, andmRNA molecules, which do not contain introns. Additional coding ornon-coding sequences may, but need not, be present within apolynucleotide of the present invention, and a polynucleotide may, butneed not, be linked to other molecules and/or support materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes a polypeptide/protein of the invention or aportion thereof) or may comprise a sequence that encodes a variant orderivative, preferably and immunogenic variant or derivative, of such asequence.

Typically, polynucleotide variants will contain one or moresubstitutions, additions, deletions and/or insertions, preferably suchthat the immunogenicity of the polypeptide encoded by the variantpolynucleotide is not substantially diminished relative to a polypeptideencoded by a polynucleotide sequence specifically set forth herein). Theterm “variants” should also be understood to encompasses homologousgenes of xenogenic origin.

In additional embodiments, the present invention provides polynucleotidefragments comprising various lengths of contiguous stretches of sequenceidentical to or complementary to one or more of the SEQ ID NO: 1 or 3.For example, polynucleotides are provided by this invention thatcomprise at least about 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300,400, the SEQ ID NO: 1 as well as all intermediate lengths there between.It will be readily understood that “intermediate lengths”, in thiscontext, means any length between the quoted values, such as 16, 17, 18,19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100,101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integersthrough 200-500; 500-1,000, and the like. Particularly preferredpolynucleotides are those which encode the epitopes as set forth in SEQID NOS: 13-94.

In another embodiment of the invention, polynucleotide compositions areprovided that are capable of hybridizing under moderate to highstringency conditions to a polynucleotide sequence provided herein, or afragment thereof, or a complementary sequence thereof. Hybridizationtechniques are well known in the art of molecular biology. For purposesof illustration, suitable moderately stringent conditions for testingthe hybridization of a polynucleotide of this invention with otherpolynucleotides include prewashing in a solution of 5×SSC, 0.5% SDS, 1.0mM EDTA (pH 8.0); hybridizing at 50° C.-60° C., 5×SSC, overnight;followed by washing twice at 65° C. for 20 minutes with each of 2×, 0.5×and 0.2×SSC containing 0.1% SDS. One skilled in the art will understandthat the stringency of hybridization can be readily manipulated, such asby altering the salt content of the hybridization solution and/or thetemperature at which the hybridization is performed. For example, inanother embodiment, suitable highly stringent hybridization conditionsinclude those described above, with the exception that the temperatureof hybridization is increased, e.g., to 60-65° C. or 65-70° C.

In certain preferred embodiments, the polynucleotides described above,e.g., polynucleotide variants, fragments and hybridizing sequences,encode polypeptides that are immunologically cross-reactive with apolypeptide sequence specifically set forth in SEQ ID NO:1 or SEQ IDNO:3. In other preferred embodiments, such polynucleotides encodepolypeptides that have a level of immunogenic activity of at least about50%, preferably at least about 70%, and more preferably at least about90% of that for a polypeptide sequence specifically set forth herein.

The polynucleotides of the present invention, or fragments thereof,regardless of the length of the coding sequence itself, may be combinedwith other DNA sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length may be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant DNA protocol. For example, illustrative polynucleotidesegments with total lengths of about 10,000, about 5000, about 3000,about 2,000, about 1,000, about 500, about 200, about 100, about 50 basepairs in length, and the like, (including all intermediate lengths) arecontemplated to be useful in many implementations of this invention.

When comparing polynucleotide sequences, two sequences are said to be“identical” if the sequence of nucleotides in the two sequences is thesame when aligned for maximum correspondence, as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W.and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA80:726-730.

Alternatively, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman (1981)Add. APL. Math 2:482, by the identity alignment algorithm of Needlemanand Wunsch (1970) J. Mol. Biol. 48:443, by the search for similaritymethods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT,BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or byinspection.

One preferred example of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al. (1977)Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol.215:403410, respectively. BLAST and BLAST 2.0 can be used, for examplewith the parameters described herein, to determine percent sequenceidentity for the polynucleotides of the invention. Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. In one illustrative example,cumulative scores can be calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments, (B) of 50, expectation (E) of 10, M=5, N=-4 and a comparisonof both strands.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12percent, as compared to the reference sequences (which does not compriseadditions or deletions) for optimal alignment of the two sequences. Thepercentage is calculated by determining the number of positions at whichthe identical nucleic acid bases occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the reference sequence (i.e., thewindow size) and multiplying the results by 100 to yield the percentageof sequence identity.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a polypeptide as described herein. Some of thesepolynucleotides bear minimal homology to the nucleotide sequence of anynative gene. Nonetheless, polynucleotides that vary due to differencesin codon usage are specifically contemplated by the present invention.Further, alleles of the genes comprising the polynucleotide sequencesprovided herein are within the scope of the present invention. Allelesare endogenous genes that are altered as a result of one or moremutations, such as deletions, additions and/or substitutions ofnucleotides. The resulting mRNA and protein may, but need not, have analtered structure or function. Alleles may be identified using standardtechniques (such as hybridization, amplification and/or databasesequence comparison).

Therefore, in another embodiment of the invention, a mutagenesisapproach, such as site-specific mutagenesis, is employed for thepreparation of immunogenic variants and/or derivatives of thepolypeptides described herein. By this approach, specific modificationsin a polypeptide sequence can be made through mutagenesis of theunderlying polynucleotides that encode them. These techniques provides astraightforward approach to prepare and test sequence variants, forexample, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into thepolynucleotide.

Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Mutations may be employed in aselected polynucleotide sequence to improve, alter, decrease, modify, orotherwise change the properties of the polynucleotide itself, and/oralter the properties, activity, composition, stability, or primarysequence of the encoded polypeptide.

In certain embodiments of the present invention, the inventorscontemplate the mutagenesis of the disclosed polynucleotide sequences toalter one or more properties of the encoded polypeptide, such as theimmunogenicity of a polypeptide vaccine. The techniques of site-specificmutagenesis are well-known in the art, and are widely used to createvariants of both polypeptides and polynucleotides. For example,site-specific mutagenesis is often used to alter a specific portion of aDNA molecule. In such embodiments, a primer comprising typically about14 to about 25 nucleotides or so in length is employed, with about 5 toabout 10 residues on both sides of the junction of the sequence beingaltered.

As will be appreciated by those of skill in the art, site-specificmutagenesis techniques have often employed a phage vector that exists inboth a single stranded and double stranded form. Typical vectors usefulin site-directed mutagenesis include vectors such as the M13 phage.These phage are readily commercially-available and their use isgenerally well-known to those skilled in the art. Double-strandedplasmids are also routinely employed in site directed mutagenesis thateliminates the step of transferring the gene of interest from a plasmidto a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double-stranded vector that includes within itssequence a DNA sequence that encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

The preparation of sequence variants of the selected peptide-encodingDNA segments using site-directed mutagenesis provides a means ofproducing potentially useful species and is not meant to be limiting asthere are other ways in which sequence variants of peptides and the DNAsequences encoding them may be obtained. For example, recombinantvectors encoding the desired peptide sequence may be treated withmutagenic agents, such as hydroxylamine, to obtain sequence variants.Specific details regarding these methods and protocols are found in theteachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991;Kuby, 1994; and Maniatis et al., 1982, each incorporated herein byreference, for that purpose.

As used herein, the term “oligonucleotide directed mutagenesisprocedure” refers to template-dependent processes and vector-mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” is intended to refer to a process that involvesthe template-dependent extension of a primer molecule. The term templatedependent process refers to nucleic acid synthesis of an RNA or a DNAmolecule wherein the sequence of the newly synthesized strand of nucleicacid is dictated by the well-known rules of complementary base pairing(see, for example, Watson, 1987). Typically, vector mediatedmethodologies involve the introduction of the nucleic acid fragment intoa DNA or RNA vector, the clonal amplification of the vector, and therecovery of the amplified nucleic acid fragment. Examples of suchmethodologies are provided by U.S. Pat. No. 4,237,224, specificallyincorporated herein by reference in its entirety.

In another approach for the production of polypeptide variants of thepresent invention, recursive sequence recombination, as described inU.S. Pat. No. 5,837,458, may be employed. In this approach, iterativecycles of recombination and screening or selection are performed to“evolve” individual polynucleotide variants of the invention having, forexample, enhanced immunogenic activity.

According to another embodiment of the present invention, polynucleotidecompositions comprising antisense oligonucleotides are provided.Antisense oligonucleotides have been demonstrated to be effective andtargeted inhibitors of protein synthesis, and, consequently, provide atherapeutic approach by which a disease can be treated by inhibiting thesynthesis of proteins that contribute to the disease. The efficacy ofantisense oligonucleotides for inhibiting protein synthesis is wellestablished. For example, the synthesis of polygalactauronase and themuscarine type 2 acetylcholine receptor are inhibited by antisenseoligonucleotides directed to their respective mRNA sequences (U.S. Pat.No. 5,739,119 and U.S. Pat. No. 5,759,829). Further, examples ofantisense inhibition have been demonstrated with the nuclear proteincyclin, the multiple drug resistance gene (MDG1), ICAM-1, E-selectin,STK-1, striatal GABAA receptor and human EGF (Jaskulski et al., Science.1988 June 10;240(4858): 1544-6; Vasanthakumar and Ahmed, Cancer Commun.1989; 1(4):225-32; Peris et al., Brain Res Mol Brain Res. 1998 June15;57(2):310-20; U.S. Pat. No. 5,801,154; U.S. Pat. No. 5,789,573; U.S.Pat. No. 5,718,709 and U.S. Pat. No. 5,610,288). Antisense constructshave also been described that inhibit and can be used to treat a varietyof abnormal cellular proliferations, e.g. cancer (U.S. Pat. No.5,747,470; U.S. Pat. No. 5,591,317 and U.S. Pat. No. 5,783,683).

Therefore, in certain embodiments, the present invention providesoligonucleotide sequences that comprise all, or a portion of, anysequence that is capable of specifically binding to polynucleotidesequence described herein, or a complement thereof. In one embodiment,the antisense oligonucleotides comprise DNA or derivatives thereof. Inanother embodiment, the oligonucleotides comprise RNA or derivativesthereof. In a third embodiment, the oligonucleotides are modified DNAscomprising a phosphorothioated modified backbone. In a fourthembodiment, the oligonucleotide sequences comprise peptide nucleic acidsor derivatives thereof. In each case, preferred compositions comprise asequence region that is complementary, and more preferablysubstantially-complementary, and even more preferably, completelycomplementary to one or more portions of polynucleotides disclosedherein.

Selection of antisense compositions specific for a given gene sequenceis based upon analysis of the chosen target sequence (i.e. in theseillustrative examples the rat and human sequences) and determination ofsecondary structure, Tm, binding energy, relative stability, andantisense compositions were selected based upon their relative inabilityto form dimers, hairpins, or other secondary structures that wouldreduce or prohibit specific binding to the target mRNA in a host cell.

Highly preferred target regions of the mRNA, are those which are at ornear the AUG translation initiation codon, and those sequences which aresubstantially complementary to 5′ regions of the mRNA. These secondarystructure analyses and target site selection considerations can beperformed, for example, using v.4 of the OLIGO primer analysis softwareand/or the BLASTN 2.0.5 algorithm software (Altschul et al., NucleicAcids Res. 1997 September 1;25(17):3389-402).

The use of an antisense delivery method employing a short peptidevector, termed MPG (27 residues), is also contemplated. The MPG peptidecontains a hydrophobic domain derived from the fusion sequence of HIVgp41 and a hydrophilic domain from the nuclear localization sequence ofSV40 T-antigen (Morris et al., Nucleic Acids Res. 1997 July15;25(14):2730-6). It has been demonstrated that several molecules ofthe MPG peptide coat the antisense oligonucleotides and can be deliveredinto cultured mammalian cells in less than 1 hour with relatively highefficiency (90%). Further, the interaction with MPG strongly increasesboth the stability of the oligonucleotide to nuclease and the ability tocross the plasma membrane.

According to another embodiment of the invention, the polynucleotidecompositions described herein are used in the design and preparation ofribozyme molecules for inhibiting expression of the tumor polypeptidesand proteins of the present invention in tumor cells. Ribozymes areRNA-protein complexes that cleave nucleic acids in a site-specificfashion. Ribozymes have specific catalytic domains that possessendonuclease activity (Kim and Cech, Proc Natl Acad Sci USA. 1987December;84(24):8788-92; Forster and Symons, Cell. 1987 April24;49(2):211-20). For example, a large number of ribozymes acceleratephosphoester transfer reactions with a high degree of specificity, oftencleaving only one of several phosphoesters in an oligonucleotidesubstrate (Cech et al., Cell. 1981 December; 27(3 Pt 2):487-96; Micheland Westhof, J Mol. Biol. 1990 December 5;216(3):585-610; Reinhold-Hurekand Shub, Nature. 1992May 14;357(6374):173-6). This specificity has beenattributed to the requirement that the substrate bind via specificbase-pairing interactions to the internal guide sequence (“IGS”) of theribozyme prior to chemical reaction.

Six basic varieties of naturally-occurring enzymatic RNAs are knownpresently. Each can catalyze the hydrolysis of RNA phosphodiester bondsin trans (and thus can cleave other RNA molecules) under physiologicalconditions. In general, enzymatic nucleic acids act by first binding toa target RNA. Such binding occurs through the target binding portion ofa enzymatic nucleic acid which is held in close proximity to anenzymatic portion of the molecule that acts to cleave the target RNA.Thus, the enzymatic nucleic acid first recognizes and then binds atarget RNA through complementary base-pairing, and once bound to thecorrect site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After an enzymatic nucleic acid hasbound and cleaved its RNA target, it is released from that RNA to searchfor another target and can repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous over manytechnologies, such as antisense technology (where a nucleic acidmolecule simply binds to a nucleic acid target to block its translation)since the concentration of ribozyme necessary to affect a therapeutictreatment is lower than that of an antisense oligonucleotide. Thisadvantage reflects the ability of the ribozyme to act enzymatically.Thus, a single ribozyme molecule is able to cleave many molecules oftarget RNA. In addition, the ribozyme is a highly specific inhibitor,with the specificity of inhibition depending not only on the basepairing mechanism of binding to the target RNA, but also on themechanism of target RNA cleavage. Single mismatches, orbase-substitutions, near the site of cleavage can completely eliminatecatalytic activity of a ribozyme. Similar mismatches in antisensemolecules do not prevent their action (Woolf et al., Proc Natl Acad SciUSA. 1992 August 15;89(16):7305-9). Thus, the specificity of action of aribozyme is greater than that of an antisense oligonucleotide bindingthe same RNA site.

The enzymatic nucleic acid molecule may be formed in a hammerhead,hairpin, a hepatitis 67 virus, group I intron or RNaseP RNA (inassociation with an RNA guide sequence) or Neurospora VS RNA motif.Examples of hammerhead motifs are described by Rossi et al. NucleicAcids Res. 1992 September 11;20(17):4559-65. Examples of hairpin motifsare described by Hampel et al. (Eur. Pat. Appl. Publ. No. EP 0360257),Hampel and Tritz, Biochemistry 1989 June 13;28(12):4929-33; Hampel etal., Nucleic Acids Res. 1990 January 25;18(2):299-304 and U.S. Pat. No.5,631,359. An example of the hepatitis δ virus motif is described byPerrotta and Been, Biochemistry. 1992 December 1;31(47):11843-52; anexample of the RNaseP motif is described by Guerrier-Takada et al.,Cell. 1983 December;35(3 Pt 2):849-57; Neurospora VS RNA ribozyme motifis described by Collins (Saville and Collins, Cell. 1990 May18;61(4):685-96; Saville and Collins, Proc Natl Acad Sci USA. 1991October 1;88(19):8826-30; Collins and Olive, Biochemistry. 1993 March23;32(11):2795-9); and an example of the Group I intron is described in(U.S. Pat. No. 4,987,071). All that is important in an enzymatic nucleicacid molecule of this invention is that it has a specific substratebinding site which is complementary to one or more of the target geneRNA regions, and that it have nucleotide sequences within or surroundingthat substrate binding site which impart an RNA cleaving activity to themolecule. Thus the ribozyme constructs need not be limited to specificmotifs mentioned herein.

Ribozymes may be designed as described in Int. Pat. Appl. Publ. No. WO93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595, each specificallyincorporated herein by reference) and synthesized to be tested in vitroand in vivo, as described. Such ribozymes can also be optimized fordelivery. While specific examples are provided, those in the art willrecognize that equivalent RNA targets in other species can be utilizedwhen necessary.

Ribozyme activity can be optimized by altering the length of theribozyme binding arms, or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases (seee.g., Int. Pat. Appl. Publ. No. WO 92/07065; Int. Pat. Appl. Publ. No.WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl.Publ. No. 92110298.4; U.S. Pat. No. 5,334,711; and Int. Pat. Appl. Publ.No. WO 94/13688, which describe various chemical modifications that canbe made to the sugar moieties of enzymatic RNA molecules), modificationswhich enhance their efficacy in cells, and removal of stem II bases toshorten RNA synthesis times and reduce chemical requirements.

Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) describes thegeneral methods for delivery of enzymatic RNA molecules. Ribozymes maybe administered to cells by a variety of methods known to those familiarto the art, including, but not restricted to, encapsulation inliposomes, by iontophoresis, or by incorporation into other vehicles,such as hydrogels, cyclodextrins, biodegradable nanocapsules, andbioadhesive microspheres. For some indications, ribozymes may bedirectly delivered ex vivo to cells or tissues with or without theaforementioned vehicles. Alternatively, the RNA/vehicle combination maybe locally delivered by direct inhalation, by direct injection or by useof a catheter, infusion pump or stent. Other routes of delivery include,but are not limited to, intravascular, intramuscular, subcutaneous orjoint injection, aerosol inhalation, oral (tablet or pill form),topical, systemic, ocular, intraperitoneal and/or intrathecal delivery.More detailed descriptions of ribozyme delivery and administration areprovided in Int. Pat. Appl. Publ. No. WO 94/02595 and Int. Pat. Appl.Publ. No. WO 93/23569, each specifically incorporated herein byreference.

Another means of accumulating high concentrations of a ribozyme(s)within cells is to incorporate the ribozyme-encoding sequences into aDNA expression vector. Transcription of the ribozyme sequences aredriven from a promoter for eukaryotic RNA polymerase I (pol I), RNApolymerase II (pol II), or RNA polymerase III (pol III). Transcriptsfrom pol III or pol III promoters will be expressed at high levels inall cells; the levels of a given pol II promoter in a given cell typewill depend on the nature of the gene regulatory sequences (enhancers,silencers, etc.) present nearby. Prokaryotic RNA polymerase promotersmay also be used, providing that the prokaryotic RNA polymerase enzymeis expressed in the appropriate cells Ribozymes expressed from suchpromoters have been shown to function in mammalian cells. Suchtranscription units can be incorporated into a variety of vectors forintroduction into mammalian cells, including but not restricted to,plasmid DNA vectors, viral DNA vectors (such as adenovirus oradeno-associated vectors), or viral RNA vectors (such as retroviral,semliki forest virus, sindbis virus vectors).

In another embodiment of the invention, peptide nucleic acids (PNAs)compositions are provided. PNA is a DNA mimic in which the nucleobasesare attached to a pseudopeptide backbone (Good and Nielsen, AntisenseNucleic Acid Drug Dev. 1997 7(4) 431-37). PNA is able to be utilized ina number methods that traditionally have used RNA or DNA. Often PNAsequences perform better in techniques than the corresponding RNA or DNAsequences and have utilities that are not inherent to RNA or DNA. Areview of PNA including methods of making, characteristics of, andmethods of using, is provided by Corey (Trends Biotechnol 1997June;15(6):224-9). As such, in certain embodiments, one may prepare PNAsequences that are complementary to one or more portions of the ACE mRNAsequence, and such PNA compositions may be used to regulate, alter,decrease, or reduce the translation of ACE-specific mRNA, and therebyalter the level of ACE activity in a host cell to which such PNAcompositions have been administered.

PNAs have 2-aminoethyl-glycine linkages replacing the normalphosphodiester backbone of DNA (Nielsen et al., Science 1991 December6;254(5037):1497-500; Hanvey et al., Science. 1992 November27;258(5087):1481-5; Hyrup and Nielsen, Bioorg Med Chem. 1996January;4(1):5-23). This chemistry has three important consequences:firstly, in contrast to DNA or phosphorothioate oligonucleotides, PNAsare neutral molecules; secondly, PNAs are achiral, which avoids the needto develop a stereoselective synthesis; and thirdly, PNA synthesis usesstandard Boc or Fmoc protocols for solid-phase peptide synthesis,although other methods, including a modified Merrifield method, havebeen used.

PNA monomers or ready-made oligomers are commercially available fromPerSeptive Biosystems (Framingham, Mass.). PNA syntheses by either Bocor Fmoc protocols are straightforward using manual or automatedprotocols (Norton et al., Bioorg Med Chem. 1995 April;3(4):437-45). Themanual protocol lends itself to the production of chemically modifiedPNAs or the simultaneous synthesis of families of closely related PNAs.

As with peptide synthesis, the success of a particular PNA synthesiswill depend on the properties of the chosen sequence. For example, whilein theory PNAs can incorporate any combination of nucleotide bases, thepresence of adjacent purines can lead to deletions of one or moreresidues in the product. In expectation of this difficulty, it issuggested that, in producing PNAs with adjacent purines, one shouldrepeat the coupling of residues likely to be added inefficiently. Thisshould be followed by the purification of PNAs by reverse-phasehigh-pressure liquid chromatography, providing yields and purity ofproduct similar to those observed during the synthesis of peptides.

Modifications of PNAs for a given application may be accomplished bycoupling amino acids during solid-phase synthesis or by attachingcompounds that contain a carboxylic acid group to the exposed N-terminalamine. Alternatively, PNAs can be modified after synthesis by couplingto an introduced lysine or cysteine. The ease with which PNAs can bemodified facilitates optimization for better solubility or for specificfunctional requirements. Once synthesized, the identity of PNAs andtheir derivatives can be confirmed by mass spectrometry. Several studieshave made and utilized modifications of PNAs (for example, Norton etal., Bioorg Med Chem. 1995 April;3(4):437-45; Petersen et al., J PeptSci. 1995 May-June;1(3):175-83; Orum et al., Biotechniques. 1995September;19(3):472-80; Footer et al., Biochemistry. 1996 August20;35(33):10673-9; Griffith et al., Nucleic Acids Res. 1995 August11;23(15):3003-8; Pardridge et al., Proc Natl Acad Sci USA. 1995 June6;92(12):5592-6; Boffa et al., Proc Natl Acad Sci USA. 1995 March14;92(6):1901-5; Gambacorti-Passerini et al., Blood. 1996 August15;88(4):1411-7; Armitage et al., Proc Natl Acad Sci USA. 1997 November11;94(23):12320-5; Seeger et al., Biotechniques. 1997September;23(3):512-7). U.S. Pat. No. 5,700,922 discusses PNA-DNA-PNAchimeric molecules and their uses in diagnostics, modulating protein inorganisms, and treatment of conditions susceptible to therapeutics.

Methods of characterizing the antisense binding properties of PNAs arediscussed in Rose (Anal Chem. 1993 December 15;65(24):3545-9) and Jensenet al. (Biochemistry. 1997 April 22;36(16):5072-7). Rose uses capillarygel electrophoresis to determine binding of PNAs to their complementaryoligonucleotide, measuring the relative binding kinetics andstoichiometry. Similar types of measurements were made by Jensen et al.using BIAcore™ technology.

Other applications of PNAs that have been described and will be apparentto the skilled artisan include use in DNA strand invasion, antisenseinhibition, mutational analysis, enhancers of transcription, nucleicacid purification, isolation of transcriptionally active genes, blockingof transcription factor binding, genome cleavage, biosensors, in situhybridization, and the like.

Polynucleotide Characterization and Expression

Polynucleotides compositions of the present invention may be preparedand/or manipulated using any of a variety of well established techniques(see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989, andother like references).

In other embodiments of the invention, polynucleotide sequences orfragments thereof which encode polypeptides of the invention, or fusionproteins or functional equivalents thereof, may be used in recombinantDNA molecules to direct expression of a polypeptide in appropriate hostcells. Due to the inherent degeneracy of the genetic code, other DNAsequences that encode substantially the same or a functionallyequivalent amino acid sequence may be produced and these sequences maybe used to clone and express a given polypeptide.

As will be understood by those of skill in the art, it may beadvantageous in some instances to produce polypeptide-encodingnucleotide sequences possessing non-naturally occurring codons. Forexample, codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producea recombinant RNA transcript having desirable properties, such as ahalf-life which is longer than that of a transcript generated from thenaturally occurring sequence.

Moreover, the polynucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterpolypeptide encoding sequences for a variety of reasons, including butnot limited to, alterations which modify the cloning, processing, and/orexpression of the gene product. For example, DNA shuffling by randomfragmentation and PCR reassembly of gene fragments and syntheticoligonucleotides may be used to engineer the nucleotide sequences. Inaddition, site-directed mutagenesis may be used to insert newrestriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, or introduce mutations, and soforth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences may be ligated to a heterologoussequence to encode a fusion protein. For example, to screen peptidelibraries for inhibitors of polypeptide activity, it may be useful toencode a chimeric protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the polypeptide-encoding sequence and theheterologous protein sequence, so that the polypeptide may be cleavedand purified away from the heterologous moiety.

Sequences encoding a desired polypeptide may be synthesized, in whole orin part, using chemical methods well known in the art (see Caruthers, M.H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T. et al.(1980) Nucl. Acids Res. Symp. Ser. 225-232). Alternatively, the proteinitself may be produced using chemical methods to synthesize the aminoacid sequence of a polypeptide, or a portion thereof. For example,peptide synthesis can be performed using various solid-phase techniques(Roberge, J. Y. et al. (1995) Science 269:202-204) and automatedsynthesis may be achieved, for example, using the ABI 431A PeptideSynthesizer (Perkin Elmer, Palo Alto, Calif.).

A newly synthesized peptide may be substantially purified by preparativehigh performance liquid chromatography (e.g., Creighton, T. (1983)Proteins, Structures and Molecular Principles, W H Freeman and Co., NewYork, N.Y.) or other comparable techniques available in the art. Thecomposition of the synthetic peptides may be confirmed by amino acidanalysis or sequencing (e.g., the Edman degradation procedure).Additionally, the amino acid sequence of a polypeptide, or any partthereof, may be altered during direct synthesis and/or combined usingchemical methods with sequences from other proteins, or any partthereof, to produce a variant polypeptide.

In order to express a desired polypeptide, the nucleotide sequencesencoding the polypeptide, or functional equivalents, may be insertedinto appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence. Methods which are well known to those skilled in theart may be used to construct expression vectors containing sequencesencoding a polypeptide of interest and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described, for example, in Sambrook,J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) CurrentProtocols in Molecular Biology, John Wiley & Sons, New York. N.Y.

A variety of expression vector/host systems may be utilized to containand express polynucleotide sequences. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of thevector—enhancers, promoters, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used. For example, when cloning in bacterialsystems, inducible promoters such as the hybrid lacZ promoter of thePBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid(Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammaliancell systems, promoters from mammalian genes or from mammalian virusesare generally preferred. If it is necessary to generate a cell line thatcontains multiple copies of the sequence encoding a polypeptide, vectorsbased on SV40 or EBV may be advantageously used with an appropriateselectable marker.

In bacterial systems, any of a number of expression vectors may beselected depending upon the use intended for the expressed polypeptide.For example, when large quantities are needed, for example for theinduction of antibodies, vectors which direct high level expression offusion proteins that are readily purified may be used. Such vectorsinclude, but are not limited to, the multifunctional E. coli cloning andexpression vectors such as BLUESCRIPT (Stratagene), in which thesequence encoding the polypeptide of interest may be ligated into thevector in frame with sequences for the amino-terminal Met and thesubsequent 7 residues of .beta.-galactosidase so that a hybrid proteinis produced; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J.Biol. Chem. 264:5503-5509); and the like. pGEX Vectors (Promega,Madison, Wis.) may also be used to express foreign polypeptides asfusion proteins with glutathione S-transferase (GST). In general, suchfusion proteins are soluble and can easily be purified from lysed cellsby adsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems may bedesigned to include heparin, thrombin, or factor XA protease cleavagesites so that the cloned polypeptide of interest can be released fromthe GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression ofsequences encoding polypeptides may be driven by any of a number ofpromoters. For example, viral promoters such as the ³⁵S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311.Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).

An insect system may also be used to express a polypeptide of interest.For example, in one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The sequencesencoding the polypeptide may be cloned into a non-essential region ofthe virus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of the polypeptide-encodingsequence will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which the polypeptide of interest may be expressed (Engelhard,E. K. et al. (1994) Proc. Natl. Acad. Sci. 91 :3224-3227).

In mammalian host cells, a number of viral-based expression systems aregenerally available. For example, in cases where an adenovirus is usedas an expression vector, sequences encoding a polypeptide of interestmay be ligated into an adenovirus transcription/translation complexconsisting of the late promoter and tripartite leader sequence.Insertion in a non-essential E1 or E3 region of the viral genome may beused to obtain a viable virus which is capable of expressing thepolypeptide in infected host cells (Logan, J. and Shenk, T. (1984) Proc.Natl. Acad. Sci. 81:3655-3659). In addition, transcription enhancers,such as the Rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding a polypeptide of interest. Suchsignals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf, D. et al.(1994) Results Probl. Cell Differ. 20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, COS, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is generally preferred. For example, cell lines which stablyexpress a polynucleotide of interest may be transformed using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may beallowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1990) Cell 22:817-23) geneswhich can be employed in tk.sup.- or aprt.sup.- cells, respectively.Also, antimetabolite, antibiotic or herbicide resistance can be used asthe basis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosides,neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14); and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51). Theuse of visible markers has gained popularity with such markers asanthocyanins, beta-glucuronidase and its substrate GUS, and luciferaseand its substrate luciferin, being widely used not only to identifytransformants, but also to quantify the amount of transient or stableprotein expression attributable to a specific vector system (Rhodes, C.A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding apolypeptide is inserted within a marker gene sequence, recombinant cellscontaining sequences can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with apolypeptide-encoding sequence under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells that contain and express a desiredpolynucleotide sequence may be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassayor immunoassay techniques which include, for example, membrane,solution, or chip based technologies for the detection and/orquantification of nucleic acid or protein.

Host cells transformed with a polynucleotide sequence of interest may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides of theinvention may be designed to contain signal sequences which directsecretion of the encoded polypeptide through a prokaryotic or eukaryoticcell membrane. Other recombinant constructions may be used to joinsequences encoding a polypeptide of interest to nucleotide sequenceencoding a polypeptide domain which will facilitate purification ofsoluble proteins. Such purification facilitating domains include, butare not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen. San Diego, Calif.) between the purificationdomain and the encoded polypeptide may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing a polypeptide of interest and a nucleic acidencoding 6 histidine residues preceding a thioredoxin or an enterokinasecleavage site. The histidine residues facilitate purification on IMIAC(immobilized metal ion affinity chromatography) as described in Porath,J. et al. (1992, Prot. Exp. Purif. 3:263-281) while the enterokinasecleavage site provides a means for purifying the desired polypeptidefrom the fusion protein. A discussion of vectors which contain fusionproteins is provided in Kroll, D. J. et al. (1993; DNA Cell Biol.12:441-453).

In addition to recombinant production methods, polypeptides of theinvention, and fragments thereof, may be produced by direct peptidesynthesis using solid-phase techniques (Merrifield J. (1963) J. Am.Chem. Soc. 85:2149-2154). Protein synthesis may be performed usingmanual techniques or by automation. Automated synthesis may be achieved,for example, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer). Alternatively, various fragments may be chemically synthesizedseparately and combined using chemical methods to produce the fulllength molecule.

T Cells Compositions

The present invention, in another aspect, provides T cells specific fora tumor polypeptide disclosed herein, or for a variant or derivativethereof. Such cells may generally be prepared in vitro or ex vivo, usingstandard procedures. For example, T cells may be isolated from bonemarrow, peripheral blood, or a fraction of bone marrow or peripheralblood of a patient, using a commercially available cell separationsystem, such as the Isolex™ System, available from Nexell Therapeutics,Inc. (Irvine, Calif.; see also U.S. Pat. No. 5,240,856; U.S. Pat. No.5,215,926; WO 89/06280; WO 91/16116 and WO 92/07243). Alternatively, Tcells may be derived from related or unrelated humans, non-humanmammals, cell lines or cultures.

T cells may be stimulated with a polypeptide, polynucleotide encoding apolypeptide and/or an antigen presenting cell (APC) that expresses sucha polypeptide. Such stimulation is performed under conditions and for atime sufficient to permit the generation of T cells that are specificfor the polypeptide of interest. Preferably, a tumor polypeptide orpolynucleotide of the invention is present within a delivery vehicle,such as a microsphere, to facilitate the generation of specific T cells.

T cells are considered to be specific for a polypeptide of the presentinvention if the T cells specifically proliferate, secrete cytokines orkill target cells coated with the polypeptide or expressing a geneencoding the polypeptide. T cell specificity may be evaluated using anyof a variety of standard techniques. For example, within a chromiumrelease assay or proliferation assay, a stimulation index of more thantwo fold increase in lysis and/or proliferation, compared to negativecontrols, indicates T cell specificity. Such assays may be performed,for example, as described in Chen et al., Cancer Res. 54:1065-1070,1994. Alternatively, detection of the proliferation of T cells may beaccomplished by a variety of known techniques. For example, T cellproliferation can be detected by measuring an increased rate of DNAsynthesis (e.g., by pulse-labeling cultures of T cells with tritiatedthymidine and measuring the amount of tritiated thymidine incorporatedinto DNA). Contact with a tumor polypeptide (100 ng/ml-100 μg/ml,preferably 200 ng/ml-25 μg/ml) for 3-7 days will typically result in atleast a two fold increase in proliferation of the T cells. Contact asdescribed above for 2-3 hours should result in activation of the Tcells, as measured using standard cytokine assays in which a two foldincrease in the level of cytokine release (e.g., TNF or IFN-γ) isindicative of T cell activation (see Coligan et al., Current Protocolsin Immunology, vol. 1, Wiley Interscience (Greene 1998)). T cells thathave been activated in response to a tumor polypeptide, polynucleotideor polypeptide-expressing APC may be CD4⁺ and/or CD8⁺. Tumorpolypeptide-specific T cells may be expanded using standard techniques.Within preferred embodiments, the T cells are derived from a patient, arelated donor or an unrelated donor, and are administered to the patientfollowing stimulation and expansion.

For therapeutic purposes, CD4⁺ or CD8⁺ T cells that proliferate inresponse to a tumor polypeptide, polynucleotide or APC can be expandedin number either in vitro or in vivo. Proliferation of such T cells invitro may be accomplished in a variety of ways. For example, the T cellscan be re-exposed to a tumor polypeptide, or a short peptidecorresponding to an immunogenic portion of such a polypeptide, with orwithout the addition of T cell growth factors, such as interleukin-2,and/or stimulator cells that synthesize a tumor polypeptide.Alternatively, one or more T cells that proliferate in the presence ofthe tumor polypeptide can be expanded in number by cloning. Methods forcloning cells are well known in the art, and include limiting dilution.

Pharmaceutical Compositions

In additional embodiments, the present invention concerns formulation ofone or more of the polynucleotide, polypeptide or T-cell disclosedherein in pharmaceutically-acceptable solutions for administration to acell or an animal, either alone, or in combination with one or moreother modalities of therapy. In particular, the present inventionconcerns, the use of cripto polynucleotides, polypeptides, fragments,fusions and variants in a pharmaceutical composition for the treatmentof tumours. In particular it is preferred that the Cripto is Cripto 1.

It will be understood that, if desired, a composition as disclosedherein may be administered in combination with other agents as well,such as, e.g., other proteins or polypeptides or variouspharmaceutically-active agents. In fact, there is virtually no limit toother components that may also be included, given that the additionalagents do not cause a significant adverse effect upon contact with thetarget cells or host tissues. The compositions may thus be deliveredalong with various other agents as required in the particular instance.Such compositions may be purified from host cells or other biologicalsources, or alternatively may be chemically synthesized as describedherein. Likewise, such compositions may further comprise substituted orderivatized RNA or DNA compositions.

Therefore, in another aspect of the present invention, pharmaceuticalcompositions are provided comprising one or more of the polynucleotide,polypeptide and/or T-cell compositions described herein in combinationwith a physiologically acceptable carrier. In certain preferredembodiments, the pharmaceutical compositions of the invention compriseimmunogenic polynucleotide and/or polypeptide compositions of theinvention for use in prophylactic and theraputic vaccine applications.Vaccine preparation is generally described in, for example, M. F. Powelland M. J. Newman, eds., “Vaccine Design (the subunit and adjuvantapproach),” Plenum Press (NY, 1995). Generally, such compositions willcomprise one or more polynucleotide and/or polypeptide compositions ofthe present invention in combination with one or more immunostimulants.

It will be apparent that any of the pharmaceutical compositionsdescribed herein can contain pharmaceutically acceptable salts of thepolynucleotides and polypeptides of the invention. Such salts can beprepared, for example, from pharmaceutically acceptable non-toxic bases,including organic bases (e.g., salts of primary, secondary and tertiaryamines and basic amino acids) and inorganic bases (e.g., sodium,potassium, lithium, ammonium, calcium and magnesium salts).

In another embodiment, illustrative immunogenic compositions, e.g.,vaccine compositions, of the present invention comprise DNA encoding oneor more of the polypeptides as described above, such that thepolypeptide is generated in situ. As noted above, the polynucleotide maybe administered within any of a variety of delivery systems known tothose of ordinary skill in the art. Indeed, numerous gene deliverytechniques are well known in the art, such as those described byRolland, Crit. Rev. Therap. Drug Canier Systems 15:143-198, 1998, andreferences cited therein. Appropriate polynucleotide expression systemswill, of course, contain the necessary regulatory DNA regulatorysequences for expression in a patient (such as a suitable promoter andterminating signal). Alternatively, bacterial delivery systems mayinvolve the administration of a bacterium (such asBacillus-Calnette-Guerrin) that expresses an immunogenic portion of thepolypeptide on its cell surface or secretes such an epitope.

Therefore, in certain embodiments, polynucleotides encoding immunogenicpolypeptides described herein are introduced into suitable mammalianhost cells for expression using any of a number of known viral-basedsystems. In one illustrative embodiment, retroviruses provide aconvenient and effective platform for gene delivery systems. A selectednucleotide sequence encoding a polypeptide of the present invention canbe inserted into a vector and packaged in retroviral particles usingtechniques known in the art. The recombinant virus can then be isolatedand delivered to a subject. A number of illustrative retroviral systemshave been described (e.g., U.S. Pat. No. 5,219,740; Miller and Rosman(1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993)Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin(1993) Cur. Opin. Genet. Develop. 3:102-109.

In addition, a number of illustrative adenovirus-based systems have alsobeen described. Unlike retroviruses which integrate into the hostgenome, adenoviruses persist extrachromosomally thus minimizing therisks associated with insertional mutagenesis (Haj-Ahmad and Graham(1986) J. Virol. 57:267-274; Bett et al. (1993) J. Virol. 67:5911-5921;Mittereder et al. (1994) Human Gene Therapy 5:717-729; Seth et al.(1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58;Berkner, K. L. (1988) BioTechniques 6:616-629; and Rich et al. (1993)Human Gene Therapy 4:461-476).

Various adeno-associated virus (AAV) vector systems have also beendeveloped for polynucleotide delivery. AAV vectors can be readilyconstructed using techniques well known in the art. See, e.g., U.S. Pat.Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070and WO 93/03769; Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996;Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539;Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Shellingand Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp.Med. 179:1867-1875.

Additional viral vectors useful for delivering the nucleic acidmolecules encoding polypeptides of the present invention by genetransfer include those derived from the pox family of viruses, such asvaccinia virus and avian poxvirus. By way of example, vaccinia virusrecombinants expressing the novel molecules can be constructed asfollows. The DNA encoding a polypeptide is first inserted into anappropriate vector so that it is adjacent to a vaccinia promoter andflanking vaccinia DNA sequences, such as the sequence encoding thymidinekinase (TK). This vector is then used to transfect cells which aresimultaneously infected with vaccinia. Homologous recombination servesto insert the vaccinia promoter plus the gene encoding the polypeptideof interest into the viral genome. The resulting TK.sup.(−) recombinantcan be selected by culturing the cells in the presence of5-bromodeoxyuridine and picking viral plaques resistant thereto.

A vaccinia-based infection/transfection system can be conveniently usedto provide for inducible, transient expression or coexpression of one ormore polypeptides described herein in host cells of an organism. In thisparticular system, cells are first infected in vitro with a vacciniavirus recombinant that encodes the bacteriophage T7 RNA polymerase. Thispolymerase displays exquisite specificity in that it only transcribestemplates bearing T7 promoters. Following infection, cells aretransfected with the polynucleotide or polynucleotides of interest,driven by a T7 promoter. The polymerase expressed in the cytoplasm fromthe vaccinia virus recombinant transcribes the transfected DNA into RNAwhich is then translated into polypeptide by the host translationalmachinery. The method provides for high level, transient, cytoplasmicproduction of large quantities of RNA and its translation products. See,e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990)87:6743-6747; Fuerst et al. Proc. Natl. Acad. Sci. USA (1986)83:8122-8126.

Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses,can also be used to deliver the coding sequences of interest.Recombinant avipox viruses, expressing immunogens from mammalianpathogens, are known to confer protective immunity when administered tonon-avian species. The use of an Avipox vector is particularly desirablein human and other mammalian species since members of the Avipox genuscan only productively replicate in susceptible avian species andtherefore are not infective in mammalian cells. Methods for producingrecombinant Avipoxviruses are known in the art and employ geneticrecombination, as described above with respect to the production ofvaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.

Any of a number of alphavirus vectors can also be used for delivery ofpolynucleotide compositions of the present invention, such as thosevectors described in U.S. Pat. Nos. 5,843,723; 6,015,686; 6,008,035 and6,015,694. Certain vectors based on Venezuelan Equine Encephalitis (VEE)can also be used, illustrative examples of which can be found in U.S.Pat. Nos. 5,505,947 and 5,643,576.

Moreover, molecular conjugate vectors, such as the adenovirus chimericvectors described in Michael et al. J. Biol. Chem. (1993) 268:6866-6869and Wagner et al. Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, canalso be used for gene delivery under the invention.

Additional illustrative information on these and other known viral-baseddelivery systems can be found, for example, in Fisher-Hoch et al., Proc.Natl. Acad. Sci. USA 86:317-321, 1989; Flexner et al., Ann. N.Y. Acad.Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat.Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No.4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner,Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434,1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994;Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502, 1993;Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir.Res. 73:1202-1207, 1993.

In another embodiment of the invention, a polynucleotide isadministered/delivered as “naked” DNA, for example as described in Ulmeret al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science259:1691-1692, 1993. The uptake of naked DNA may be increased by coatingthe DNA onto biodegradable beads, which are efficiently transported intothe cells.

In still another embodiment, a composition of the present invention canbe delivered via a particle bombardment approach, many of which havebeen described. In one illustrative example, gas-driven particleacceleration can be achieved with devices such as those manufactured byPowderject Pharmaceuticals PLC (Oxford, UK) and Powderject Vaccines Inc.(Madison, Wis.), some examples of which are described in U.S. Pat. Nos.5,846,796; 6,010,478; 5,865,796; 5,584,807; and EP Patent No. 0500 799.This approach offers a needle-free delivery approach wherein a drypowder formulation of microscopic particles, such as polynucleotide orpolypeptide particles, are accelerated to high speed within a helium gasjet generated by a hand held device, propelling the particles into atarget tissue of interest. The particles, when delivering nucleic acidare preferably gold beads of a 0.4-4.0 um, more preferably 0.6-2.0 umdiameter and the DNA conjugate coated onto these and then encased in acartridge for placing into the “gene gun”. The particles are typicallyand preferably delivered to the skin. Other means of delivery to theskin, comprise utilising needle delivery via a needle of a liquidformulation.

DNA vaccines usually consist of a bacterial plasmid vector into which isinserted a strong, normally viral, promoter, the gene of interest whichencodes for an antigenic peptide and a polyadenylation/transcriptionaltermination sequences. Thus gene of interest may encode a full criptoprotein as described or simply an antigenic peptide sequence such asdescribed in seq ID no 13-94. The plasmid can be grown in bacteria, suchas for example E. coli and then isolated and prepared in an appropriatemedium, depending upon the intended route of administration, beforebeing administered to the host. Following administration the plasmid istaken up by cells of the host where the encoded peptide is produced. Theplasmid vector will preferably be made without an origin of replicationwhich is functional in eukaryotic cells, in order to prevent plasmidreplication in the mammalian host and integration within chromosomal DNAof the animal concerned.

There are a number of advantages of DNA vaccination relative totraditional vaccination techniques. First, it is predicted that becauseof the proteins which are encoded by the DNA sequence are synthesised inthe host, the structure or conformation of the protein will be similarto the native protein associated with the disease state. It is alsolikely that DNA vaccination will offer protection against differentstrains of a virus, by generating cytotoxic T lymphocyte response thatrecognise epitopes from conserved proteins. Furthermore, because theplasmids are taken up by the host cells where antigenic protein can beproduced, a long-lasting immune response will be elicited. Thetechnology also offers the possibility of combing diverseimmunogens/epitopes into a single preparation.

Helpful background information in relation to DNA vaccination isprovided in Donnelly et al “DNA vaccines” Ann. Rev Immunol. 1997 15:617-648, the disclosure of which is included herein in its entirety byway of reference.

In a related embodiment, other devices and methods that may be usefulfor gas-driven needle-less injection of compositions of the presentinvention include those provided by Bioject, Inc. (Portland, Oreg.),some examples of which are described in U.S. Pat. Nos. 4,790,824;5,064,413; 5,312,335; 5,383,851; 5,399,163; 5,520,639 and 5,993,412.

According to another embodiment, the pharmaceutical compositionsdescribed herein will comprise one or more immunostimulants in additionto the immunogenic polynucleotide, polypeptide, T-cell and/or APCcompositions of this invention. An immunostimulant refers to essentiallyany substance that enhances or potentiates an immune response (antibodyand/or cell-mediated) to an exogenous antigen. One preferred type ofimmunostimulant comprises an adjuvant. Many adjuvants contain asubstance designed to protect the antigen from rapid catabolism, such asaluminum hydroxide or mineral oil, and a stimulator of immune responses,such as lipid A, Bordatella peitussis or Mycobacterium tuberculosisderived proteins. Certain adjuvants are commercially available as, forexample, Freund's Incomplete Adjuvant and Complete Adjuvant (DifcoLaboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company,Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.);aluminum salts such as aluminum hydroxide gel (alum) or aluminumphosphate; salts of calcium, iron or zinc; an insoluble suspension ofacylated tyrosine; acylated sugars; cationically or anionicallyderivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A and quil A. Cytokines, such asGM-CSF, interleukin-2, -7, -12, and other like growth factors, may alsobe used as adjuvants.

Within certain embodiments of the invention, the adjuvant composition ispreferably one that induces an immune response predominantly of the Th1type. High levels of Th1-type cytokines (e.g., IFN-γ, TNFα, IL-2 andIL-12) tend to favor the induction of cell mediated immune responses toan administered antigen. In contrast, high levels of Th2-type cytokines(e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction ofhumoral immune responses. Following application of a vaccine as providedherein, a patient will support an immune response that includes Th1- andTh2-type responses. Within a preferred embodiment, in which a responseis predominantly Th1-type, the level of Th1-type cytokines will increaseto a greater extent than the level of Th2-type cytokines. The levels ofthese cytokines may be readily assessed using standard assays. For areview of the families of cytokines, see Mosmann and Coffman, Anti. Rev.Immunol. 7:145-173, 1989.

Certain preferred adjuvants for eliciting a predominantly Th1-typeresponse include, for example, a combination of monophosphoryl lipid A,preferably 3-de-O-acylated monophosphoryl lipid A, together with analuminum salt. MPL® adjuvants are available from Corixa Corporation(Seattle, Wash.; see, for example, U.S. Pat. Nos. 4,436,727; 4,877,611;4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which theCpG dinucleotide is unmethylated) also induce a predominantly Th1response. Such oligonucleotides are well known and are described, forexample, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and5,856,462. Immunostimulatory DNA sequences are also described, forexample, by Sato et al., Science 273:352, 1996. Another preferredadjuvant comprises a saponin, such as Quil A, or derivatives thereof,including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham,Mass.); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins.Other preferred formulations include more than one saponin in theadjuvant combinations of the present invention, for example combinationsof at least two of the following group comprising QS21, QS7, Quil A,β-escin, or digitonin.

Alternatively the saponin formulations may be combined with vaccinevehicles composed of chitosan or other polycationic polymers,polylactide and polylactide-co-glycolide particles, poly-N-acetylglucosamine-based polymer matrix, particles composed of polysaccharidesor chemically modified polysaccharides, liposomes and lipid-basedparticles, particles composed of glycerol monoesters, etc. The saponinsmay also be formulated in the presence of cholesterol to formparticulate structures such as liposomes or ISCOMs. Furthermore, thesaponins may be formulated together with a polyoxyethylene ether orester, in either a non-particulate solution or suspension, or in aparticulate structure such as a paucilamelar liposome or ISCOM. Thesaponins may also be formulated with excipients such as Carbopol^(R) toincrease viscosity, or may be formulated in a dry powder form with apowder excipient such as lactose.

In one preferred embodiment, the adjuvant system includes thecombination of a monophosphoryl lipid A and a saponin derivative, suchas the combination of QS21 and 3D-MPL® adjuvant, as described in WO94/00153, or a less reactogenic composition where the QS21 is quenchedwith cholesterol, as described in WO 96/33739. Other preferredformulations comprise an oil-in-water emulsion and tocopherol. Anotherparticularly preferred adjuvant formulation employing QS21, 3D-MPL®adjuvant and tocopherol in an oil-in-water emulsion is described in WO95/17210.

Another enhanced adjuvant system involves the combination of aCpG-containing oligonucleotide and a saponin derivative particularly thecombination of CpG and QS21 as disclosed in WO 00/09159. Preferably theformulation additionally comprises an oil in water emulsion andtocopherol.

Additional illustrative adjuvants for use in the pharmaceuticalcompositions of the invention include Montanide ISA 720 (Seppic,France), SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59(Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4,available from SmithKline Beecham, Rixensart, Belgium), Detox(Enhanzyn®) (Corixa, Hamilton, Mont.), RC-529 (Corixa, Hamilton, Mont.)and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as thosedescribed in pending U.S. patent application Ser. Nos. 08/853,826 and09/074,720, the disclosures of which are incorporated herein byreference in their entireties, and polyoxyethylene ether adjuvants suchas those described in WO 99/52549A1.

Other preferred adjuvants include adjuvant molecules of the generalformula (I):HO(CH₂CH₂O)_(n)-A-R

Wherein, n is 1-50, A is a bond or —C(O)—, R is C₁₋₅₀ alkyl or PhenylC₁₋₅₀ alkyl.

One embodiment of the present invention consists of a vaccineformulation comprising a polyoxyethylene ether of general formula (I),wherein n is between 1 and 50, preferably 4-24, most preferably 9; the Rcomponent is C₁₋₅₀, preferably C₄-C₂₀ alkyl and most preferably C₁₂alkyl, and A is a bond. The concentration of the polyoxyethylene ethersshould be in the range 0.1-20%, preferably from 0.1-10%, and mostpreferably in the range 0.1-1%. Preferred polyoxyethylene ethers areselected from the following group: polyoxyethylene-9-lauryl ether,polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether,polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, andpolyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such aspolyoxyethylene lauryl ether are described in the Merck index (12^(th)edition: entry 7717). These adjuvant molecules are described in WO99/52549.

The polyoxyethylene ether according to the general formula (I) abovemay, if desired, be combined with another adjuvant. For example, apreferred adjuvant combination is preferably with CpG as described inthe pending UK patent application GB 9820956.2.

According to another embodiment of this invention, an immunogeniccomposition described herein is delivered to a host via antigenpresenting cells (APCs), such as dendritic cells, macrophages, B cells,monocytes and other cells that may be engineered to be efficient APCs.Such cells may, but need not, be genetically modified to increase thecapacity for presenting the antigen, to improve activation and/ormaintenance of the T cell response, to have anti-tumor effects per seand/or to be immunologically compatible with the receiver (i.e., matchedHLA haplotype). APCs may generally be isolated from any of a variety ofbiological fluids and organs, including tumor and peritumoral tissues,and may be autologous, allogeneic, syngeneic or xenogeneic cells.

Certain preferred embodiments of the present invention use dendriticcells or progenitors thereof as antigen-presenting cells. Dendriticcells are highly potent APCs (Banchereau and Steinman, Nature392:245-251, 1998) and have been shown to be effective as aphysiological adjuvant for eliciting prophylactic or therapeuticantitumor immunity (see Timmerman and Levy, Ann. Rev. Med. 50:507-529,1999). In general, dendritic cells may be identified based on theirtypical shape (stellate in situ, with marked cytoplasmic processes(dendrites) visible in vitro), their ability to take up, process andpresent antigens with high efficiency and their ability to activatenaive T cell responses. Dendritic cells may, of course, be engineered toexpress specific cell-surface receptors or ligands that are not commonlyfound on dendritic cells in vivo or ex vivo, and such modified dendriticcells are contemplated by the present invention. As an alternative todendritic cells, secreted vesicles antigen-loaded dendritic cells(called exosomes) may be used within a vaccine (see Zitvogel et al.,Nature Med. 4:594-600, 1998).

Dendritic cells and progenitors may be obtained from peripheral blood,bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltratingcells, lymph nodes, spleen, skin, umbilical cord blood or any othersuitable tissue or fluid. For example, dendritic cells may bedifferentiated ex vivo by adding a combination of cytokines such asGM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytes harvested fromperipheral blood. Alternatively, CD34 positive cells harvested fromperipheral blood, umbilical cord blood or bone marrow may bedifferentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce differentiation, maturation andproliferation of dendritic cells.

Dendritic cells are conveniently categorized as “immature” and “mature”cells, which allows a simple way to discriminate between two wellcharacterized phenotypes. However, this nomenclature should not beconstrued to exclude all possible intermediate stages ofdifferentiation. Immature dendritic cells are characterized as APC witha high capacity for antigen uptake and processing, which correlates withthe high expression of Fcy receptor and mannose receptor. The maturephenotype is typically characterized by a lower expression of thesemarkers, but a high expression of cell surface molecules responsible forT cell activation such as class I and class II MHC, adhesion molecules(e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80,CD86 and 4-1BB).

APCs may generally be transfected with a polynucleotide of the invention(or portion or other variant thereof) such that the encoded polypeptide,or an immunogenic portion thereof, is expressed on the cell surface.Such transfection may take place ex vivo, and a pharmaceuticalcomposition comprising such transfected cells may then be used fortherapeutic purposes, as described herein. Alternatively, a genedelivery vehicle that targets a dendritic or other antigen presentingcell may be administered to a patient, resulting in transfection thatoccurs in vivo. In vivo and ex vivo transfection of dendritic cells, forexample, may generally be performed using any methods known in the art,such as those described in WO 97/24447, or the gene gun approachdescribed by Mahvi et al., Immunology and cell Biology 75:456460, 1997.Antigen loading of dendritic cells may be achieved by incubatingdendritic cells or progenitor cells with the tumor polypeptide, DNA(naked or within a plasmid vector) or RNA; or with antigen-expressingrecombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus orlentivirus vectors). Prior to loading, the polypeptide may be covalentlyconjugated to an immunological partner that provides T cell help (e.g.,a carrier molecule). Alternatively, a dendritic cell may be pulsed witha non-conjugated immunological partner, separately or in the presence ofthe polypeptide.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will typically vary depending on the mode ofadministration. Compositions of the present invention may be formulatedfor any appropriate manner of administration, including for example,topical, oral, nasal, mucosal, intravenous, intracranial,intraperitoneal, subcutaneous and intramuscular administration.

Carriers for use within such pharmaceutical compositions arebiocompatible, and may also be biodegradable. In certain embodiments,the formulation preferably provides a relatively constant level ofactive component release. In other embodiments, however, a more rapidrate of release immediately upon administration may be desired. Theformulation of such compositions is well within the level of ordinaryskill in the art using known techniques. Illustrative carriers useful inthis regard include microparticles of poly(lactide-co-glycolide),polyacrylate, latex, starch, cellulose, dextran and the like. Otherillustrative delayed-release carriers include supramolecular biovectors,which comprise a non-liquid hydrophilic core (e.g., a cross-linkedpolysaccharide or oligosaccharide) and, optionally, an external layercomprising an amphiphilic compound, such as a phospholipid (see e.g.,U.S. Pat. No. 5,151,254 and PCT applications WO 94/20078, WO/94/23701and WO 96/06638). The amount of active compound contained within asustained release formulation depends upon the site of implantation, therate and expected duration of release and the nature of the condition tobe treated or prevented.

In another illustrative embodiment, biodegradable microspheres (e.g.,polylactate polyglycolate) are employed as carriers for the compositionsof this invention. Suitable biodegradable microspheres are disclosed,for example, in U.S. Pat. Nos. 4,897,268; 5,075,109; 5,928,647;5,811,128; 5,820,883; 5,853,763; 5,814,344, 5,407,609 and 5,942,252.Modified hepatitis B core protein carrier systems, such as described inWO/99 40934, and references cited therein, will also be useful for manyapplications. Another illustrative carrier/delivery system employs acarrier comprising particulate-protein complexes, such as thosedescribed in U.S. Pat. No. 5,928,647, which are capable of inducing aclass I-restricted cytotoxic T lymphocyte responses in a host.

The pharmaceutical compositions of the invention will often furthercomprise one or more buffers (e.g., neutral buffered saline or phosphatebuffered saline), carbohydrates (e.g., glucose, mannose, sucrose ordextrans), mannitol, proteins, polypeptides or amino acids such asglycine, antioxidants, bacteriostats, chelating agents such as EDTA orglutathione, adjuvants (e.g., aluminum hydroxide), solutes that renderthe formulation isotonic, hypotonic or weakly hypertonic with the bloodof a recipient, suspending agents, thickening agents and/orpreservatives. Alternatively, compositions of the present invention maybe formulated as a lyophilizate.

The pharmaceutical compositions described herein may be presented inunit-dose or multi-dose containers, such as sealed ampoules or vials.Such containers are typically sealed in such a way to preserve thesterility and stability of the formulation until use. In general,formulations may be stored as suspensions, solutions or emulsions inoily or aqueous vehicles. Alternatively, a pharmaceutical compositionmay be stored in a freeze-dried condition requiring only the addition ofa sterile liquid carrier immediately prior to use.

The development of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation, is well known in the art,some of which are briefly discussed below for general purposes ofillustration.

In certain applications, the pharmaceutical compositions disclosedherein may be delivered via oral administration to an animal. As such,these compositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

The active compounds may even be incorporated with excipients and usedin the form of ingestible tablets, buccal tables, troches, capsules,elixirs, suspensions, syrups, wafers, and the like (see, for example,Mathiowitz et al., Nature 1997 March 27;386(6623):410-4; Hwang et al.,Crit Rev Ther Drug Carrier Syst 1998;15(3):243-84; U.S. Pat. No.5,641,515; U.S. Pat. No. 5,580,579 and U.S. Pat. No. 5,792,451).Tablets, troches, pills, capsules and the like may also contain any of avariety of additional components, for example, a binder, such as gumtragacanth, acacia, cornstarch, or gelatin; excipients, such asdicalcium phosphate; a disintegrating agent, such as corn starch, potatostarch, alginic acid and the like; a lubricant, such as magnesiumstearate; and a sweetening agent, such as sucrose, lactose or saccharinmay be added or a flavoring agent, such as peppermint, oil ofwintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar, or both.Of course, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compounds may be incorporated intosustained-release preparation and formulations.

Typically, these formulations will contain at least about 0.1% of theactive compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 60% or 70% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

For oral administration the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. Alternatively, the active ingredientmay be incorporated into an oral solution such as one containing sodiumborate, glycerin and potassium bicarbonate, or dispersed in adentifrice, or added in a therapeutically-effective amount to acomposition that may include water, binders, abrasives, flavoringagents, foaming agents, and humectants. Alternatively the compositionsmay be fashioned into a tablet or solution form that may be placed underthe tongue or otherwise dissolved in the mouth.

In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein parenterally,intravenously, intramuscularly, or even intraperitoneally orintradermally. Such approaches are well known to the skilled artisan,some of which are further described, for example, in U.S. Pat. No.5,543,158; U.S. Pat. No. 5,641,515 and U.S. Pat. No. 5,399,363. Incertain embodiments, solutions of the active compounds as free base orpharmacologically acceptable salts may be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions mayalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations generally will contain a preservative to prevent the growthof microorganisms.

Illustrative pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (for example, see U.S. Pat. No. 5,466,468). In all cases theform must be sterile and must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, and/or vegetable oils.Proper fluidity may be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and/or by the use of surfactants. The preventionof the action of microorganisms can be facilitated by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

In one embodiment, for parenteral administration in an aqueous solution,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. Moreover, for human administration, preparationswill of course preferably meet sterility, pyrogenicity, and the generalsafety and purity standards as required by FDA Office of Biologicsstandards.

In another embodiment of the invention, the compositions disclosedherein may be formulated in a neutral or salt form. Illustrativepharmaceutically-acceptable salts include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective.

The carriers can further comprise any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions. The phrase “pharmaceutically-acceptable” refersto molecular entities and compositions that do not produce an allergicor similar untoward reaction when administered to a human.

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering genes, nucleic acids, and peptidecompositions directly to the lungs via nasal aerosol sprays has beendescribed, e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No. 5,804,212.Likewise, the delivery of drugs using intranasal microparticle resins(Takenaga et al., J Controlled Release 1998 March 2;52(1-2):81-7) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871) are alsowell-known in the pharmaceutical arts. Likewise, illustrativetransmucosal drug delivery in the form of a polytetrafluoroetheylenesupport matrix is described in U.S. Pat. No. 5,780,045.

In certain embodiments, liposomes, nanocapsules, microparticles, lipidparticles, vesicles, and the like, are used for the introduction of thecompositions of the present invention into suitable hostcells/organisms. In particular, the compositions of the presentinvention may be formulated for delivery either encapsulated in a lipidparticle, a liposome, a vesicle, a nanosphere, or a nanoparticle or thelike. Alternatively, compositions of the present invention can be bound,either covalently or non-covalently, to the surface of such carriervehicles.

The formation and use of liposome and liposome-like preparations aspotential drug carriers is generally known to those of skill in the art(see for example, Lasic, Trends Biotechnol 1998 July;16(7):307-21;Takakura, Nippon Rinsho 1998 March;56(3):691-5; Chandran et al., IndianJ Exp Biol. 1997August;35(8):801-9; Margalit, Crit Rev Ther Drug CarrierSyst. 1995;12(2-3):233-61; U.S. Pat. No. 5,567,434; U.S. Pat. No.5,552,157; U.S. Pat. No. 5,565,213; U.S. Pat. No. 5,738,868 and U.S.Pat. No. 5,795,587, each specifically incorporated herein by referencein its entirety).

Liposomes have been used successfully with a number of cell types thatare normally difficult to transfect by other procedures, including Tcell suspensions, primary hepatocyte cultures and PC 12 cells (Remeisenet al., J Biol. Chem. 1990 September 25;265(27):1633742; Muller et al.,DNA Cell Biol. 1990 April 9(3):221-9). In addition, liposomes are freeof the DNA length constraints that are typical of viral-based deliverysystems. Liposomes have been used effectively to introduce genes,various drugs, radiotherapeutic agents, enzymes, viruses, transcriptionfactors, allosteric effectors and the like, into a variety of culturedcell lines and animals. Furthermore, he use of liposomes does not appearto be associated with autoimmune responses or unacceptable toxicityafter systemic delivery.

In certain embodiments, liposomes are formed from phospholipids that aredispersed in an aqueous medium and spontaneously form multilamellarconcentric bilayer vesicles (also termed multilamellar vesicles (MLVs).

Alternatively, in other embodiments, the invention provides forpharmaceutically-acceptable nanocapsule formulations of the compositionsof the present invention. Nanocapsules can generally entrap compounds ina stable and reproducible way (see, for example, Quintanar-Guerrero etal., Drug Dev Ind Pharm. 1998 December;24(12):1113-28). To avoid sideeffects due to intracellular polymeric overloading, such ultrafineparticles (sized around 0.1 μm) may be designed using polymers able tobe degraded in vivo. Such particles can be made as described, forexample, by Couvreur et al., Crit Rev Ther Drug Carrier Syst.1988;5(1):1-20; zur Muhlen et al., Eur J Pharm Biopharm. 1998March;45(2):149-55; Zambaux et al. J Controlled Release. 1998 January 250(1-3):3140; and U.S. Pat. No. 5,145,684.

Cancer Therapeutic Methods

In further aspects of the present invention, the pharmaceuticalcompositions described herein may be used for the treatment of cancer,particularly for the immunotherapy of colon or colorectal cancer. Inother embodiments the compositions can be used to treat breast, ornon-small cell lung carcinoma. Typically the composition will be usefulfor treating patients whose cancers express cripto antigen and/or whosemetastases express cripto antigen. Within such methods, thepharmaceutical compositions described herein are administered to apatient, typically a warm-blooded animal, preferably a human. A patientmay or may not be afflicted with cancer. Accordingly, the abovepharmaceutical compositions may be used to prevent the development of acancer or to treat a patient afflicted with a cancer. Pharmaceuticalcompositions and vaccines may be administered either prior to orfollowing surgical removal of primary tumors and/or treatment such asadministration of radiotherapy or conventional chemotherapeutic drugs.As discussed above, administration of the pharmaceutical compositionsmay be by any suitable method, including administration by intravenous,intraperitoneal, intramuscular, subcutaneous, intranasal, intradermal,anal, vaginal, topical and oral routes.

Within certain embodiments, immunotherapy may be active immunotherapy,in which treatment relies on the in vivo stimulation of the endogenoushost immune system to react against tumors with the administration ofimmune response-modifying agents (such as polypeptides andpolynucleotides as provided herein).

Within other embodiments, immunotherapy may be passive immunotherapy, inwhich treatment involves the delivery of agents with establishedtumor-immune reactivity (such as effector cells) that can directly orindirectly mediate antitumor effects and does not necessarily depend onan intact host immune system. Examples of effector cells include T cellsas discussed above, T lymphocytes (such as CD8+ cytotoxic T lymphocytesand CD4+ T-helper tumor-infiltrating lymphocytes), killer cells (such asNatural Killer cells and lymphokine-activated killer cells), B cells andantigen-presenting cells (such as dendritic cells and macrophages)expressing a polypeptide provided herein. T cell receptors and antibodyreceptors specific for the polypeptides recited herein may be cloned,expressed and transferred into other vectors or effector cells foradoptive immunotherapy.

Effector cells may generally be obtained in sufficient quantities foradoptive immunotherapy by growth in vitro, as described herein. Cultureconditions for expanding single antigen-specific effector cells toseveral billion in number with retention of antigen recognition in vivoare well known in the art. Such in vitro culture conditions typicallyuse intermittent stimulation with antigen, often in the presence ofcytokines (such as IL-2) and non-dividing feeder cells. As noted above,immunoreactive polypeptides as provided herein may be used to rapidlyexpand antigen-specific T cell cultures in order to generate asufficient number of cells for immunotherapy. In particular,antigen-presenting cells, such as dendritic, macrophage, monocyte,fibroblast and/or B cells, may be pulsed with immunoreactivepolypeptides or transfected with one or more polynucleotides usingstandard techniques well known in the art. For example,antigen-presenting cells can be transfected with a polynucleotide havinga promoter appropriate for increasing expression in a recombinant virusor other expression system. Cultured effector cells for use in therapymust be able to grow and distribute widely, and to survive long term invivo. Studies have shown that cultured effector cells can be induced togrow in vivo and to survive long term in substantial numbers by repeatedstimulation with antigen supplemented with IL-2 (see, for example,Cheever et al., Immunological Reviews 157:177, 1997).

Alternatively, a vector expressing a polypeptide recited herein may beintroduced into antigen presenting cells taken from a patient andclonally propagated ex vivo for transplant back into the same patient.Transfected cells may be reintroduced into the patient using any meansknown in the art, preferably in sterile form by intravenous,intracavitary, intraperitoneal or intratumor administration.

Routes and frequency of administration of the therapeutic compositionsdescribed herein, as well as dosage, will vary from individual toindividual, and may be readily established using standard techniques. Ingeneral, the pharmaceutical compositions and vaccines may beadministered by injection (e.g., intracutaneous, intramuscular,intravenous, intradermally or subcutaneous), intranasally (e.g., byaspiration) or orally. Preferably, between 1 and 10 doses may beadministered over a 52 week period. Preferably, 6 doses areadministered, at intervals of 1 month, and booster vaccinations may begiven periodically thereafter. Alternate protocols may be appropriatefor individual patients. A suitable dose is an amount of a compoundthat, when administered as described above, is capable of promoting ananti-tumor immune response, and is at least 10-50% above the basal(i.e., untreated) level. Such response can be monitored by measuring theanti-tumor antibodies in a patient or by vaccine-dependent generation ofcytolytic effector cells capable of killing the patient's tumor cells invitro. Such vaccines should also be capable of causing an immuneresponse that leads to an improved clinical outcome (e.g., more frequentremissions, complete or partial or longer disease-free survival) invaccinated patients as compared to non-vaccinated patients. In general,for pharmaceutical compositions and vaccines comprising one or morepolypeptides, the amount of each polypeptide present in a dose rangesfrom about 25 μg to 5 mg per kg of host. Suitable dose sizes will varywith the size of the patient, but will typically range from about 0.1 mlto about 5 ml.

In general, an appropriate dosage and treatment regimen provides theactive compound(s) in an amount sufficient to provide therapeutic and/orprophylactic benefit. Such a response can be monitored by establishingan improved clinical outcome (e.g., more frequent remissions, completeor partial, or longer disease-free survival) in treated patients ascompared to non-treated patients. Increases in preexisting immuneresponses to a tumor protein generally correlate with an improvedclinical outcome. Such immune responses may generally be evaluated usingstandard proliferation, cytotoxicity or cytokine assays, which may beperformed using samples obtained from a patient before and aftertreatment.

EXAMPLE 1a

Expression of Immunoreactive Cripto-1 in Human Lesions

Non-involved Premalignant Tissue Epithelium Lesion Carcinoma TA TVAColon 25/193 (13) 26/65(40) 10/13 (77) 122/168 (73)** IM  17/37 (46)**Stomach  1/37 (3) 16/30 (53) Pancreas 10/58 (17)  58/98 (59)**Hyperplasias Adenomas Gall Bladder N.D. 6/9(67) 4/7(58)  89/132 (68)**DCIS Breast  5/33 (15) 26/55 (47) 497/631 (79)** Non Small 178/195(91)** Cell Lung Non- involved Cystadenomas Tissue Epithelium SerousMucinous Serous Mucinous Ovary 6/7 (86) 6/14 0/7 4/10 (40)  4/5 (80)post- (43) menopausal 23/40 (58) 25/48 (52) 3/9 (33) 3/8 (38) 4/8 (50) 9/10 (90) Borderline: 10/10 Endo- 10/28 (36) 53/91 (58)* metrium post-menopausal Hyperplasias 18/30 (60) 68/96 (71)* Cervix 4/25 (17) 40/74(54)* Testis 0/3 29/51 (57)** Embryonal Carcinomas Seminomas 19/19(100)** 10/32 (31) Adrenal  1/3 (33) Cortex Bladder 0/6 23/39 (60)**Renal  0/18 Prostate  0/9 TA = Tubular adenoma TVC = Tubulovillousadenoma IM = Intestinal metaplasia **Statistically significant expressonin carcinomas over non-involved tissues

EXAMPLES EXAMPLE 1.6 Over-Expression of Cripto in Cancerous Tissues

Real-time RT-PCR (U. Gibson. 1996. Genome Research: 6,996) is used tocompare mRNA transcript abundance of the target protein in a panel ofnormal and tumor tissues and/or cell lines. This analysis is critical toestablish the tumor specificity of Cripto expression, which is animportant criterion a good vaccine candidate must fulfil.

Total RNA is extracted from snap frozen biopsies or cell lines usingTriPure reagent (Roche). Total RNA from normal tissues is also purchasedfrom InVitrogen. Poly-A+ mRNA is purified from total RNA after DNAasetreatment using oligo-dT magnetic beads (Dynal). Quantification of themRNA is performed by spectrofluorimetry (VersaFluor, BioRad) usingRiboGreen dye (Molecular Probes).

TaqMan primers (forward primer sequence: TGGGTAGGAAAGAGGAAGCAAAT, SEQ IDNO:7; reverse primer sequence: TGCTTCTCTACCACCACCTAATCA, SEQ ID NO:8)and probe for real-time RT-PCR amplification are designed with thePerkin-Elmer Primer Express software using default options for TaqManamplification conditions.

Real-time reactions are assembled according to standard PCR protocolsusing 2 ng of reverse transcribed mRNA (Expand RT, Roche) for eachreaction. Either SybrI or TaqMan detection is undergone, depending onthe evaluated sample. In case of SybrI detection, SybrI dye (MolecularProbes) is added at a final dilution of 1/75000 for real-time detection,and TaqMan probe is omitted. Amplification (40 cycles) and real-timedetection is performed in a Perkin-Elmer Biosystems PE7700 system usingconventional instrument settings. Ct values are calculated using thePE7700 Sequence Detector Software. Ct values are obtained from eachtissue sample for the target mRNA (CtX) and for the actin mRNA (CtA).

As the efficiency of PCR amplification under the prevailing experimentalconditions is close to the theoretical amplification efficiency,2^((CtA-CtX)) value is an estimate of the relative target transcriptlevel of the sample, standardized with respect to Actin transcriptlevel. A value of 1 thus suggests the candidate antigen and Actin havethe same expression level.

RT-PCR analysis, using SybrI detection, was performed on a set of colontumor and matched normal colon from 6 different patients and 48 normaltissue samples. A TaqMan detection was run on a set of colon tumor andmatched normal colon from 6 other patients (reactions were run intriplicates) and 48 normal tissue samples. Tested normal tissues (andthe abbreviations used in graphics) are shown below:

-   -   Adrenal gland (Ad_GI)    -   Aorta (Ao)    -   Bladder (Bl)    -   Bonne marrow Bo_Ma    -   Brain (Bra, Bra1, Bra2, Bra3, Bra4, Bra5)    -   Cervix (Ce)    -   Colon (Co)    -   Fallopian tube (Fa_Tu)    -   Heart (He)    -   Ileum (I1)    -   Kidney (Ki)    -   Liver (Li, Li1, Li2)    -   Lung (Lu)    -   Lymph node (Ly_No)    -   Esophagus (Oe)    -   Ovary (Ov)    -   Pancreas (Pa, Panc1, Panc2)    -   Parathyrois (Pa_Thy)    -   Placenta (Pl)    -   Prostate (Pr)    -   Rectum (Re)    -   Skin (Sk)    -   Skeletal muscle (Sk_Mu)    -   Small intestine (Sm_In)    -   Spleen (Sp)    -   Stomach (St)    -   Testis (Te)    -   Thyroid (Thyr, Thy, Thy1, Thy2)    -   Thymus (Thym1,) Thym2    -   Trachea (Tr, Tra)

Real-time RT-PCR reactions, using Sybri detection, were also performedon a set of 7 lung cell lines:

-   -   CRL-5803 (Carcinoma, Non-Small Cell Lung Cancer, large cell,        neuroendocrine, metastatic site: lymph node)    -   CRL-5807 (Bronchioalveolar carcinoma, Non-Small Cell Lung        Cancer)    -   CRL-5810 (Adenocarcinoma, Non-Small Cell Lung Cancer)    -   CRL-5815 (Carcinoid, lung bronchus)    -   CRL-5865 (Adenocarcinoma, metastatic site: pleural effusion)    -   CRL-9609 (Normal lung, bronchus, epithelial, virus transformed)    -   HTB-177 (Carcinoma, large cell lung cancer, pleural effusion)        and 2 colon cell lines:    -   CRL-2159 (Carcinoma, Cecum, Dukes' B)    -   CCL-250

Fresh biopsy normal lung tissues (Lu(ucl), Lu(IVG)) and a lung tumortissue (LuTum) were also performed as control.

RT-PCR results on colorectal biopsies and normal tissues are shown inFIGS. 1, 2, 3 and 4, and in Table 1. RT-PCR results on cell lines areshown in FIG. 5.

TABLE 1 Cripto expression in colorectal tumors and normal tissues. Sybrdetection¹ TaqMan detection¹ Colorectal tumor versus adjacent normalcolon² Number of over-expressing 5/6 5/6 patients Averageover-expression fold 200 90 in over-expressing patients Medianover-expression fold 64 (22-724) 20 (4-397) (minimum-maximum) Colorectaltumor versus average normal tissues² Number of over-expressing 5/6 4/6patients Average over-expression fold 10 12 in over-expressing patientsMedian over-expression fold 11 (3-22) 7 (3-32) (minimum-maximum) Normaltissues with a high Spleen (0.5) Spleen (0.75), ovary transcript level³(normal-to- (2)⁴ tumor ratio) ¹Transcript levels were calculated incolorectal tumors and a panel of normal tissues using 2 detectiontechniques: TaqMan and Sybr. Regarding Cripto, TaqMan detection involved6 patients and measures were done in triplicates, whereas Sybr detectionwas undergone on 6 different patients. ²Transcript level in colorectaltumors was compared to both matched normal colon and average of normaltissue transcript levels. ³A normal tissue has a high transcript levelwhen it is higher than one fifth of colorectal tumors transcript level.⁴Ovary has not been evaluated in Cripto Sybr experiment.

Table 1, FIGS. 1, 2, 3 & 4 clearly show that Cripto, while beingmarginally expressed in normal adult tissues, is highly over-expressedin a majority of colorectal tumors, with an over-expression rate of morethan ten fold. Moreover, FIG. 5 indicates Cripto is dramaticallyover-expressed in a lung tumor cell line (CRL-5815). Cripto tumorassociated antigen is therefore a suitable vaccine candidate to treatboth colorectal and lung cancer patients.

EXAMPLE 2 Cloning of Cripto-1 c-DNA from Lung Tumor Cell Lines

Total RNA was extracted using TriPure reagent from 10⁷ cultured cells of7 different lung cell lines (see section 1 for the complete list of celllines). Total RNAs were pooled, and mRNA was purified from pooled totalRNA on oligo-d(T) magnetic beads (Dynal). 250 ng of mRNA were used forcDNA synthesis. Quantification of the mRNA is performed byspectrofluorimetry (VersaFluor, BioRad) using RiboGreen dye (MolecularProbes). cDNA was synthesized using the GeneRacer technology(Invitrogen) which ensures the amplification of only full-lengthtranscripts. mRNA was treated with CIP. mRNA 5′ ends were decapped withTAP (Tobacco Acid Pyrophosphatase) and were ligated to a specific RNAoligonucleotide. The ligated mRNA was reverse transcribed into cDNAusing an oligod(T)-tailed primer. Amplification of cDNA was performedusing both GeneRacer flanking primers (Advantage, Clontech). Criptoamplification was performed on 10 ng of GeneRacer cDNA using genespecific PCR primers (forward primer sequence:CGTCCAAGGCCGAAAGCCCTCCAGTT, SEQ ID NO:9; and reverse primer sequence:TTGGGAGAGGGCAGGGCAAAGAAGTAAGAA, SEQ ID NO:10). PCR reaction was donewith Advantage II Taq DNA polymerase (Clontech) under standardconditions. PCR product was cloned in pCR4-TOPO plasmid (Invitrogen).Amplified sequence (SEQ ID NO:95) was shown to display a variation atcodon 22 (SEQ ID NO:96): Ala (GCC) instead of Val (GTC) in the nativeversion (SeqID6). Native version was restored by PCR mutagenesis.

EXAMPLE 3 Immunogenicity of Cripto Tumor-Associated Antigen in AnimalModels

The immunogenicity of the antigen of the present invention can beverified by immunizing rabbits and mice using various means ofimmunization. Indeed, immunization with Cripto forms, either peptide orrecombinant protein could induce humoral immune response with thegeneration of specific antibodies against Cripto and/or could induce aCripto specific cellular immune response. In vivo delivery of Criptoprotein using for instance, naked DNA in an appropriate vector encodingCripto or fragments of Cripto, Cripto genes delivered by a viral vectorencoding Cripto or fragments of Critpo, could also be useful todemonstrate Cripto immunogenicity.

3.1: Synthetic Peptide Immunization.

The synthetic peptides from human Cripto-1 amino-acid sequence selectedto immunize rabbits are GHQEFARPSRGYL (13 amino acids, SEQ ID NO:11),and QEEPAIRPRSSQRVPPMG (18 amino acids, SEQ ID NO:12). Syntheticpeptides are then conjugated to a carrier protein (KLH). Conjugates areformulated with Freund's adjuvant, and two rabbits are immunized witheach of the conjugates. Four weeks after the second immunization andfour weeks after the third immunization, a blood sample is taken.Anti-Cripto antibody titers are estimated in the serum by ELISA and/orWestern Blot following standard protocols (see section 3.5).

3.2: Nucleic Acids Immunization.

pcDNA3.1 vector (Invitrogen) is used to construct the vaccinatingplasmid. To promote secretion of the in vivo translated protein and totherefore induce the humoral response against the present inventionantigen, nucleic acid sequence encoding Cripto-1 with its own signalpeptide is inserted into the vector polycloning site. The recombinantexpression plasmid is used to transform a host E. coli strain such asBL21.

The above recombinant strain is grown in conventional cell culturemedium. Bacteria are harvested before reaching the stationary phase.Plasmid preparation using Quiagen system is undergone for injection inmice.

Six to eight weeks-old Balb/c mice receive intramuscular injections ofrecombinant expression plasmid. Two weeks after the last injection, ablood sample is taken. Titers of specific antibodies elicited againstthe present invention antigen are determined by ELISA and/or WesternBlot (see section 3.5).

3.3 Viral Vector Immunization Using Adenoviruses.

Recombinant adenoviruses are effective vectors for gene-basedvaccination because they are capable of eliciting humoral and cellularimmune responses against the encoded antigen. The nucleotide sequencecoding for Cripto-1 protein with its own signal sequence could beinserted in an appropriated Adeno derivative viral vector. Theadenoviral recombinant vector could be administrated to mice bydifferent routes (intramuscular, intranasal, intradermal, subcutaneousor intraeperitoneal) After two week, blood samples could be taken andthe titer of antibodies elicited examined. Additional experiments tomeasure the cellular immune response could also be performed.

3.4: Recombinant Protein Immunization.

3.4.1: Expression and Purification of Cripto-1 Recombinant Protein

Expression in microbial hosts, is used to produce the whole protein orfragments of the invention antigen for immunization purposes.Recombinant proteins may be expressed in two microbial hosts, E. coliand in yeast (such as Saccharomyces cerevisiae or Pichia pastoris). Thisallows the selection of the expression system with the best features forthis particular antigen production.

The expression strategy first involves the design of the primarystructure of the recombinant antigen. In general, an expression fusionpartner (EFP) to improve levels of expression and/or an immune fusionpartner (IFP) to modulate the immunogenic properties of the antigen, areplaced at the N-terminal extremity. In addition, an affinity fusionpartner (AFP) useful for facilitating further purification is includedat the C-terminal end.

When the recombinant strains are available, the recombinant product ischaracterized by the evaluation of the level of expression and theprediction of further solubility of the engineered protein by analysisof its behavior in the crude extract.

After growth in appropriate culture medium and induction of therecombinant protein expression, total extracts are analyzed by SDS-PAGE.The recombinant proteins are visualized in stained gels and identifiedby Western blot analysis using the specific anti-peptide antibodiesgenerated by peptide immunization in rabbit (see section 3.1).

A comparative evaluation of the different versions of the expressedantigen and expression hosts will allow the selection of the mostpromising candidate and host that is to be used for further purificationand further immunological evaluation.

The purification schemes follow a classical approach based on thepresence of a Histidine affinity tail in the recombinant protein. In atypical experiment the disrupted cells are filtered and the acellularextracts loaded onto an Ion Metal Affinity Chromatography (IMAC; Ni++NTAfrom Qiagen) that will specifically retain the recombinant protein. Theretained proteins are eluted by 0-500 mM Imidazole gradient (possibly inpresence of a detergent) in a phosphate buffer.

3.4.2: Protein immunization

Rabbits are immunized, intramuscularly several times at several weekintervals with recombinant purified protein, formulated in the adjuvant3D-MPL/QS21. Three weeks after each immunization, blood samples aretaken. Anti-Cripto antibody titer is estimated in the serum by ELISA.The specificity of the anti-Cripto antibodies generated is tested byWestern Blot (see section 3.5) using the purified protein and includingappropriated controls.

3.5: Immunological Response Assays of Cripto-Immunized Animals.

Humoral response to Cripto immunization is assessed by measuring Criptospecific antibody titers in animal sera using ELISA and Western Blot.The following material harboring Cripto-1 derived peptides or fullprotein could be used for such test:

-   -   Cripto synthetic peptides (see section 3.1 for possible        peptides), or    -   protein extracts from cultures of Cripto-expressing cell lines        (see section 1 for possible cell lines), or    -   lysates of COS cells that have been engineered to transiently        express a Cripto recombinant plasmid (see below), or    -   protein extracts of recombinant E. coli or yeast strains (see        section 3.3.1 for recombinant strain generation), or    -   purified recombinant antigen (see section 3.3.1 for antigen        purification).

Transient expression of Cripto in COS cells is obtained by transientlytransfecting COS cells with recombinant pcDNA3.1 plasmid prepared fornucleic acid vaccination (see section 3.2).

For ELISA reactions, one of the above mentioned antigen is coated onmicrotiter plates (purified recombinant protein is preferred). ELISA isthen performed using standard protocol.

Western Blots are realized under standard conditions with one of theabove mentioned antigen (purified recombinant protein is preferred,synthetic peptides are not used)

The cellular response can also be assessed by stimulating in vitrovaccinated mouse spleen cells with the peptides used to immunize themice, or with antigen-derived overlapping peptides covering the wholeantigen sequence.

EXAMPLE 4 Demonstration of Existing Cripto Specific Human T-Cell by InVitro Priming

Immunological relevance of Cripto-1 can be further confirmed by in vitropriming of human T cells. All T cell lymphocytes, T cell lines anddendritic cells are derived from PBMCs (peripheral blood mononuclearcells) of healthy donors or cancer patients (preferred donors are fromHLA-A2 subtype).

Epitopes Binding of HLA Alleles Prediction:

The HLA Class I binding peptide sequences (nonamers, decamers) arepredicted either by the Parker's algorithm [Parker K, et al. 1994](available on the world wide web at tbimas.dcrt.nih.gov/molibio/hlabind/) and the Rammensee method [Rammensee, et al. 1997] [Rammensee, etal. 1995] (available on the world wide web atsyfpeithi.bmi-h-eldelberg.com/Scripts/MHCServer.dll/EpPredict.htm).

The HLA Class II binding peptide sequences (nonamers) are predictedusing the Tepitope algorithm [Sturniolo, et al. 1999].

CD8+ T-Cell Response:

Two strategies to raise the CD8+ T cell lines are used: a peptide-basedapproach and a whole gene-based approach. Both approaches require thefull-length cDNA of interest in the correct reading frame to be clonedin an appropriate delivery system and to be used to predict the sequenceof the HLA binding peptides.

Peptide-Based Approach:

For this approach, an HLA-A2.1/Kb transgenic mouse model is used forscreening of the HLA-A2.1 peptides. Briefly, transgenic mice areimmunized with adjuvanted HLA-A2 peptides, those able to induce a CD8response (as defined by an efficient lysis or g-IFN production onpeptide-pulsed target cells) are further analyzed in the human system.

Human dendritic cells (cultured according to [Romani et al.]) will bepulsed with the selected peptides and used to stimulate CD8+-sorted Tcells (by Facs). After several weekly stimulation, the CD8+lines arefirst tested on peptide-pulsed autologous BLCL (EBV-B transformed celllines). To verify the proper in vivo processing of the peptide, the CD8+lines are then tested on cDNA-transfected tumor cells (HLA-A2transfected LnCaP, Skov3 or CAMA tumor cells).

Whole Gene-Based Approach:

CD8+ T cell lines are primed and stimulated with either gene-guntransfected dendritic cells, retrovirally transduced B7.1-transfectedfibroblasts, recombinant pox virus [Kim et al.] or adenovirus[Butterfield et al.] infected dendritic cells. Virus infected cells arevery efficient to present antigenic peptides since the antigen isexpressed at high level but can only be used once to avoid theover-growth of viral T cells lines.

After alternated stimulation, the CD8+ lines are tested oncDNA-transfected tumor cells as indicated above. Peptide specificity andidentity is determined to confirm the immunological validation ofantigen of the present invention.

CD4+ T-Cell Response:

Similarly, the CD4+ T-cell immune response can also be assessed.Generation of specific CD4+ T cells is made using dendritic cells loadedwith recombinant purified protein or peptides to stimulate the T-cells.

Results:

A—Prediction of Class I Epitopes Using the Parker Method.

A-1 Cripto-1 and -3 Class I Epitopes.

HLA Epitope Start SEQ ID allele size position Sequence Score NO: A68Decamer 12 SVIWIMAISK 240.000 SEQ ID NO:13 A68 Decamer 117 SVPHDTWLPK120.000 SEQ ID NO:14 B2705 Nonamer 33 HQEFARPSR 100.000 SEQ ID NO:15B2705 Nonamer 59 IRPRSSQRV 600.000 SEQ ID NO:16 B2705 Nonamer 72IQHSKELNR 100.000 SEQ ID NO:17 B2705 Nonamer 103 GRNCEHDVR 1000.000 SEQID NO:18 B2705 Nonamer 110 VRKENCGSV 600.000 SEQ ID NO:19 B2705 Nonamer138 LRCFPQAFL 2000.000 SEQ ID NO:20 B2705 Nonamer 161 SRTPELPPS 200.000SEQ ID NO:21 B2705 Decamer 65 QRVPPMGIQH 200.000 SEQ ID NO:22 B2705Decamer 79 NRTCCLNGGT 200.000 SEQ ID NO:23 B2705 Decamer 103 GRNCEHDVRK2000.000 SEQ ID NO:93 B2705 Decamer 136 GQLRCFPQAF 100.000 SEQ ID NO:24B2705 Decamer 161 SRTPELPPSA 200.000 SEQ ID NO:94 B5101 Decamer 143QAFLPGCDGL 110.000 SEQ ID NO:25 B5102 Decamer 143 QAFLPGCDGL 302.500 SEQID NO:25 B5102 Decamer 150 DGLVMDEHLV 120.000 SEQ ID NO:26 B60 Nonamer23 FELGLVAGL 325.000 SEQ ID NO:27 B60 Nonamer 76 KELNRTCCL 320.000 SEQID NO:28 B62 Nonamer 137 QLRCFPQAF 240.000 SEQ ID NO:29 B62 Decamer 136GQLRCFPQAF 160.000 SEQ ID NO:24  B7 Nonamer 169 SARTTTFML 120.000 SEQ IDNO:30A-2 Cripto-1 Specific Class I Epitopes.

HLA Epitope Start SEQ ID allele size position Sequence Score NO: A0201Nonamer 5 KMARFSYSV 668.086 SEQ ID NO:31 A0201 Decamer 16 IMAISKVFEL349.885 SEQ ID NO:32 A0201 Decamer 13 VIWIMAISKV 310.361 SEQ ID NO:33A0201 Decamer 175 FMLVGICLSI 128.242 SEQ ID NO:34 B2702 Nonamer 7ARFSYSVIW 500.000 SEQ ID NO:35 B2702 Nonamer 3 CRKMARFSY 200.000 SEQ IDNO:36 B2702 Decamer 7 ARFSYSVIWI 300.000 SEQ ID NO:37 B2702 Decamer 37ARPSRGYLAF 200.000 SEQ ID NO:38 B2705 Nonamer 7 ARFSYSVIW 1000.000 SEQID NO:35 B2705 Nonamer 3 CRKMARFSY 1000.000 SEQ ID NO:36 B2705 Nonamer170 ARTTTFMLV 600.000 SEQ ID NO:39 B2705 Nonamer 37 ARPSRGYLA 200.000SEQ ID NO:40 B2705 Decamer 7 ARFSYSVIWI 3000.000 SEQ ID NO:37 B2705Decamer 37 ARPSRGYLAF 1000.000 SEQ ID NO:38 B2705 Decamer 3 CRKMARFSYS200.000 SEQ ID NO:41 B4403 Decamer 34 QEFARPSRGY 120.000 SEQ ID NO:42B5101 Nonamer 6 MARFSYSVI 286.000 SEQ ID NO:43 B5101 Decamer 169SARTTTFMLV 110.000 SEQ ID NO:44 B5102 Nonamer 17 MAISKVFEL 150.000 SEQID NO:45 B5102 Nonamer 6 MARFSYSVI 100.000 SEQ ID NO:43 B5103 Nonamer 6MARFSYSVI 100.000 SEQ ID NO:43 B5103 Decamer 169 SARTTTFMLV 121.000 SEQID NO:44 B7 Nonamer 36 FARPSRGYL 180.000 SEQ ID NO:46A-3 Cripto-3 Specific Class I Epitopes.

HLA Epitope Start SEQ ID allele size position Sequence Score NO: A0201Nonamer 5 KMVRFSYSV 668.086 SEQ ID NO:47 A0201 Nonamer 89 CMLESFCAC103.417 SEQ ID NO:48 A0201 Decamer 16 IMAISKAFEL 152.124 SEQ ID NO:49A0201 Decamer 175 FMLAGICLSI 128.242 SEQ ID NO:50 B2702 Nonamer 7VRFSYSVIW 500.000 SEQ ID NO:51 B2702 Nonamer 3 CRKMVRFSY 200.000 SEQ IDNO:52 B2702 Decamer 7 VRFSYSVIWI 300.000 SEQ ID NO:53 B2702 Decamer 37ARPSRGDLAF 200.000 SEQ ID NO:54 B2705 Nonamer 3 CRKMVRFSY 1000.000 SEQID NO:52 B2705 Nonamer 7 VRFSYSVIW 1000.000 SEQ ID NO:51 B2705 Nonamer37 ARPSRGDLA 200.000 SEQ ID NO:55 B2705 Nonamer 170 ARTTTFMLA 200.000SEQ ID NO:56 B2705 Decamer 7 VRFSYSVIWI 3000.000 SEQ ID NO:53 B2705Decamer 37 ARPSRGDLAF 1000.000 SEQ ID NO:54 B2705 Decamer 59 IRPRSSQRVL600.000 SEQ ID NO:57 B2705 Decamer 3 CRKMVRFSYS 200.000 SEQ ID NO:58B2705 Decamer 65 QRVLPMGIQH 200.000 SEQ ID NO:59 B5101 Nonamer 60RPRSSQRVL 120.000 SEQ ID NO:60 B5102 Nonamer 17 MAISKAFEL 150.000 SEQ IDNO:61 B7 Nonamer 60 RPRSSQRVL 800.000 SEQ ID NO:60 B7 Nonamer 36FARPSRGDL 180.000 SEQ ID NO:62

NB: Score is an estimate of half-time of disassociation of a moleculecontaining this subsequence.

B—Prediction of Class I Epitopes Using the Rammensee Method.

B-1 Cripto-1 and -3 Class I Epitopes.

HLA Epitope Start SEQ ID allele size position Sequence Score NO: A26Nonamer 179 GICLSIQSY 27 SEQ ID NO:63 A26 Decamer 27 LVAGLGHQE 25 SEQ IDF NO:64 A26 Decamer 121 DTWLPKKCSL 25 SEQ ID NO:65 A3  Nonamer 58AIRPRSSQR 29 SEQ ID NO:66 A3  Nonamer 13 VIWIMAISK 24 SEQ ID NO:67 A3 Decamer 12 SVIWIMAISK 29 SEQ ID NO:13 A3  Decamer 117 SVPHDTWLP 24 SEQID K NO:14 B2705 Nonamer 103 GRNCEHDVR 25 SEQ ID NO:18 B2705 Nonamer 138LRCFPQAFL 24 SEQ ID NO:20B-2 Cripto-1 Specific Class I Epitopes.

HLA Epitope Start SEQ ID allele size position Sequence Score NO: A0201Nonamer 83 CLNGGTCML 27 SEQ ID NO:68 A0201 Nonamer 5 KMARFSYSV 25 SEQ IDNO:31 A0201 Decamer 16 IMAISKVFEL 28 SEQ ID NO:32 A0201 Decamer 13VIWINAISKV 26 SEQ ID NO:33 A26 Nonamer 35 EFARPSRGY 24 SEQ ID NO:69  A3Nonamer 66 RVPPMGIQH 27 SEQ ID NO:70  A3 Nonamer 21 KVFELGLVA 24 SEQ IDNO:71 B08 Nonamer 17 MAISKVFEL 26 SEQ ID NO:45 B5101 Nonamer 6 MARFSYSVI25 SEQ ID NO:43B-3 Cripto-3 Specific Class I Epitopes.

HLA Epitope Start SEQ ID allele size position Sequence Score NO: A0201Nonamer 83 CLNGGTCML 27 SEQ ID NO:68 A0201 Nonamer 176 MLAGICLSI 25 SEQID NO:72 A0201 Decamer 16 IMAISKAFEL 24 SEQ ID NO:49  A3 Nonamer 66RVLPMGIQH 29 SEQ ID NO:73 B0702 Nonamer 60 RPRSSQRVL 24 SEQ ID NO:60 B08Nonamer 17 MAISKAFEL 25 SEQ ID NO:61C—Prediction of Class II Epitopes Using the Tepitope Method.C-1 Cripto-1 and -3 Class II Epitopes.

HLA Epitope Start SEQ ID allele size position Sequence Score NO:DRB1*0102 Nonamer 70 MGIQHSKEL 1.8 SEQ ID NO:74 DRB1*0301 Nonamer 152LVMDEHLVA 5.9 SEQ ID NO:75 DRB1*0301 Nonamer 158 LVASRTPEL 4.3 SEQ IDNO:76 DRB1*0401 Nonamer 152 LVMDEHLVA 3.2 SEQ ID NO:75 DRB1*0402 Nonamer59 IRPRSSQRV 4.9 SEQ ID NO:16 DRB1*0703 Nonamer 11 YSVIWIMAI 6 SEQ IDNO:77 DRB1*0703 Nonamer 158 LVASRTPEL 5.7 SEQ ID NO:76 DRB1*0802 Nonamer59 IRPRSSQRV 1.8 SEQ ID NO:16 DRB1*0802 Nonamer 123 WLPKKCSLC 2.3 SEQ IDNO:78 DRB1*0804 Nonamer 59 IRPRSSQRV 2.8 SEQ ID NO:16 DRB1*0806 Nonamer59 IRPRSSQRV 3.1 SEQ ID NO:16 DRB1*1101 Nonamer 11 YSVIWIMAI 2.8 SEQ IDNO:77 DRB1*1101 Nonamer 152 LVMDEHLVA 2.2 SEQ ID NO:75 DRB1*1102 Nonamer152 LVMDEHLVA 2.4 SEQ ID NO:75 DRB1*1104 Nonamer 25 LGLVAGLGH 2.8 SEQ IDNO:79 DRB1*1104 Nonamer 152 LVMDEHLVA 3.2 SEQ ID NO:75 DRB1*1106 Nonamer25 LGLVAGLGH 2.8 SEQ ID NO:79 DRB1*1106 Nonamer 152 LVMDEHLVA 3.2 SEQ IDNO:75 DRB1*1107 Nonamer 46 FRDDSIWPQ 2.8 SEQ ID NO:80 DRB1*1107 Nonamer152 LVMDEHLVA 5.9 SEQ ID NO:75 DRB1*1107 Nonamer 158 LVASRTPEL 3.3 SEQID NO:76 DRB1*1305 Nonamer 11 YSVIWIMAI 3.7 SEQ ID NO:77 DRB1*1307Nonamer 11 YSVIWIMAI 1.2 SEQ ID NO:77 DRB1*1501 Nonamer 138 LRCFPQAFL4.3 SEQ ID NO:20 DRB1*1501 Nonamer 152 LVMDEHLVA 4.5 SEQ ID NO:75DRB1*1502 Nonamer 152 LVMDEHLVA 3.5 SEQ ID NO:75 DRB5*0101 Nonamer 13VIWIMAISK 5.3 SEQ ID NO:67C-2 Cripto-1 Specific Class H Epitopes.

HLA Epitope Sequence SEQ ID allele size position Start Score NO:DRB1*0101 Nonamer 15 WIMAISKVF 1.3 SEQ ID NO:81 DRB1*0102 Nonamer 176MLVGICLSI 1.8 SEQ ID NO:82 DRB1*0102 Nonamer 178 VGICLSIQS 1.7 SEQ IDNO:83 DRB1*0301 Nonamer 17 MAISKVFEL 3.9 SEQ ID NO:45 DRB1*0401 Nonamer178 VGICLSIQS 2.8 SEQ ID NO:83 DRB1*0402 Nonamer 178 VGICLSIQS 4.2 SEQID NO:83 DRB1*0404 Nonamer 14 IWIMAISKV 2.9 SEQ ID NO:84 DRB1*0404Nonamer 177 LVGICLSIQ 3.3 SEQ ID NO:85 DRB1*0404 Nonamer 178 VGICLSIQS3.8 SEQ ID NO:83 DRB1*0405 Nonamer 175 FMLVGICLS 3.2 SEQ ID NO:86DRB1*0405 Nonamer 177 LVGICLSIQ 3.1 SEQ ID NO:85 DRB1*0405 Nonamer 178VGICLSIQS 2.8 SEQ ID NO:83 DRB1*0703 Nonamer 15 WIMAISKVF 5.7 SEQ IDNO:81 DRB1*0703 Nonamer 17 NAISKVFEL 7.6 SEQ ID NO:45 DRB1*0703 Nonamer176 MLVGICLSI 5 SEQ ID NO:82 DRB1*0801 Nonamer 175 FMLVGICLS 3.8 SEQ IDNO:86 DRB1*0802 Nonamer 175 FMLVGICLS 3.8 SEQ ID NO:86 DRB1*0804 Nonamer175 FMLVGICLS 2.8 SEQ ID NO:86 DRB1*1101 Nonamer 175 FMLVGICLS 3.9 SEQID NO:86 DRB1*1101 Nonamer 178 VGICLSIQS 2.4 SEQ ID NO:83 DRB1*1104Nonamer 175 FMLVGICLS 2.9 SEQ ID NO:86 DRB1*1104 Nonamer 177 LVGICLSIQ2.6 SEQ ID NO:85 DRB1*1104 Nonamer 178 VGICLSIQS 3.4 SEQ ID NO:83DRB1*1106 Nonamer 175 FMLVGICLS 2.9 SEQ ID NO:86 DRB1*1106 Nonamer 177LVGICLSIQ 2.6 SEQ ID NO:85 DRB1*1106 Nonamer 178 VGICLSIQS 3.4 SEQ IDNO:83 DRB1*1107 Nonamer 17 MAISKVFEL 2.9 SEQ ID NO:45 DRB1*1107 Nonamer177 LVGICLSIQ 3.6 SEQ ID NO:85 DRB1*1107 Nonamer 178 VGICLSIQS 3 SEQ IDNO:83 DRB1*1302 Nonamer 15 WINAISKVF 3.3 SEQ ID NO:81 DRB1*1302 Nonamer175 FMLVGICLS 3 SEQ ID NO:86 DRB1*1305 Nonamer 15 WIMAISKVF 3.1 SEQ IDNO:81 DRB1*1305 Nonamer 175 FMLVGICLS 4.3 SEQ ID NO:86 DRB1*1307 Nonamer175 FMLVGICLS 3.9 SEQ ID NO:86 DRB1*1307 Nonamer 177 LVGICLSIQ 1.6 SEQID NO:85 DRB1*1501 Nonamer 6 MARFSYSVI 4.5 SEQ ID NO:43 DRB1*1501Nonamer 176 MLVGICLSI 4.1 SEQ ID NO:82 DRB1*1502 Nonamer 6 MARFSYSVI 3.5SEQ ID NO:43 DRB5*0101 Nonamer 15 WIMAISKVF 4.1 SEQ ID NO:81C-2 Cripto-3 Specific Class II Epitopes.

HLA Epitope Start SEQ ID allele size position Sequence Score NO:DRB1*0101 Nonamer 15 WIMAISKAF 1.3 SEQ ID NO:87 DRB1*0102 Nonamer 6MVRFSYSVI 1.4 SEQ ID NO:88 DRB1*0102 Nonamer 17 MAISKAFEL 1:6 SEQ IDNO:61 DRB1*0401 Nonamer 7 VRFSYSVIW 2.9 SEQ ID NO:51 DRB1*0401 Nonamer175 FMLAGICLS 2.7 SEQ ID NO:89 DRB1*0402 Nonamer 67 VLPMGIQHS 3.6 SEQ IDNO:90 DRB1*0404 Nonamer 6 MVRFSYSVI 2.5 SEQ ID NO:88 DRB1*0404 Nonamer14 IWIMAISKA 3.6 SEQ ID NO:91 DRB1*0404 Nonamer 67 VLPMGIQHS 3.5 SEQ IDNO:90 DRB1*0405 Nonamer 175 FMLAGICLS 2.7 SEQ ID NO:89 DRB1*0703 Nonamer7 VRFSYSVIW 6.5 SEQ ID NO:51 DRB1*0703 Nonamer 15 WIMAISKAF 5.7 SEQ IDNO:87 DRB1*0703 Nonamer 17 MAISKAFEL 7.5 SEQ ID NO:61 DRB1*0801 Nonamer16 IMAISKAFE 3 SEQ ID NO:92 DRB1*0801 Nonamer 175 FMLAGICLS 3.5 SEQ IDNO:89 DRB1*0802 Nonamer 67 VLPMGIQHS 1.9 SEQ ID NO:90 DRB1*0802 Nonamer175 EMLAGICLS 3.5 SEQ ID NO:89 DRB1*0804 Nonamer 67 VLPMGIQHS 2.9 SEQ IDNO:90 DRB1*0804 Nonamer 175 FMLAGICLS 2.5 SEQ ID NO:89 DRB1*0806 Nonamer16 IMAISKAFE 4 SEQ ID NO:92 DRB1*1101 Nonamer 175 FMLAGICLS 3.5 SEQ IDNO:89 DRB1*1102 Nonamer 67 VLPMGIQHS 3.1 SEQ ID NO:90 DRB1*1102 Nonamer175 FMLAGICLS 2.5 SEQ ID NO:89 DRB1*1107 Nonamer 7 VRFSYSVIW 2.8 SEQ IDNO:51 DRB1*1301 Nonamer 67 VLPMGIQHS 3.5 SEQ ID NO:90 DRB1*1302 Nonamer15 WIMAISKAF 3.3 SEQ ID NO:87 DRB1*1302 Nonamer 175 FMLAGICLS 3.9 SEQ IDNO:89 DRB1*1305 Nonamer 15 WIMAISKAF 3.1 SEQ ID NO:87 DRB1*1305 Nonamer175 FMLAGICLS 3.9 SEQ ID NO:89 DRB1*1307 Nonamer 67 VLPMGIQRS 1.5 SEQ IDNO:90 DRB1*1307 Nonamer 175 FMLAGICLS 3.5 SEQ ID NO:89 DRB1*1501 Nonamer6 MVRPSYSVI 6.6 SEQ ID NO:88 DRB1*1502 Nonamer 6 MVRFSYSVI 5.6 SEQ IDNO:88 DRB5*0101 Nonamer 15 WIMAISKAF 4.1 SEQ ID NO:87

EXAMPLE 5 Anti-Tumor Potential of the Humoral Response Induced by CriptoVaccines

A series of experiments aimed at assessing the inhibitory effect of theCripto-specific humoral response on the pathophysiological activities ofCripto in cancer could be carried out by using various standards invitro assays. The in vitro assays could be, but not restricted to,growth inhibition assay, cell motility inhibition assay, chemotaxisinhibition assay, inhibition of the invasion through extracellularmatrix protein (ECM), and growth signal transduction pathway inhibition.The effects of the sera of immunized animals with Cripto peptides orprotein in adjuvant, or with a plasmid DNA or a viral delivery system,e.g. adenoviral vector, encoding the Cripto protein could be assessed inthese in vitro assays that gauge Cripto-mediated biological effects onCripto-expressing cell lines. In parallel, the effect of the pre-immunesera from the same animals will be tested as negative control. The celllines used in these assays could be, but not limited to, human tumorcells expressing Cripto such as GEO cells, NTERA2 cells, the CRL-5815cell line, or murine tumor cell lines that naturally over-expressCripto. As example, the inhibition of the in vitro growth of both GEOand NTERA2 cells has previously been demonstrated by treatment withanti-sense oligonucleotides designed to prevent the translation ofCripto mRNA [Baldassarre et al., 1996; Ciardiello et al., 1994; Alper etal., 2000].

5.1: Immunization Protocol:

Mice or rabbits would be immunized on day 0, 14, and 21 by intra-footpadinjections of either peptides or protein in adjuvant, intra-dermalinjections of a plasmid DNA encoding Cripto using gene-gun devices, orintra-dermal injections of a viral vector delivery system, e.g.adenoviral vector, encoding Cripto.

5.2: Cell Proliferation Inhibition Assay:

The sera from immunized animals will be collected and added at differentdilutions to the culture medium of cells platted in 96-well plates.Similarly, pre-immune sera of these animals will also be added to cellsas negative control. The cells will be treated for 3 to 7 days. The cellgrowth will be measured by standard methods such as ³H-thymidineincorporation assay, MTT assay, crystal violet staining, or colonycounting for proliferation in soft agar-medium.

5.3: Cell Invasion, Motility, and Chemotaxis Inhibition Assays:

The sera from immunized animals will be collected pre- andpost-immunization and added at different dilutions to cell suspensionsused for invasion, motility, and chemotaxis standards assays. Theinhibition of Cripto-mediated invasion, motility, and chemotaxis couldbe assessed with the use of, for instance, but limited to, commerciallyavailable Falcon chambers with matrigel inserts (CollaborativeResearch), agarose droplet motility assay [Yamamoto et al., 1990], andcommercially available Boyden apparatus (Neuro Probe), respectively.

5.4: Signal Transduction Inhibition Assays:

The sera from immunized animals will be collected pre- andpost-immunization and added at different dilutions to Cripto-expressingtumor cells in culture. Then, exogenous Cripto protein or human sera ormilk containing the highest concentration of Cripto detected by ELISA[Bianco et al., 2001] will be added to the culture medium of the cells.After various incubation times, the cells will be harvested andprocessed by standard methods in order to perform immunoblot analysis.The phosphorylation status of various key signal transduction pathwaysinvolved in cell proliferation will be assessed in theseCripto-simulated cells in the presence of pre- or post-immune sera. Forinstance, the tyrosine phosphorlation status of erb B-4 andmitogen-activated protein (MAP) kinase family such as ERK1, ERK-2, andP38 will be assessed by immunoblotting with antibodies that recognizethe phosphorylated, active form of these enzymes as previous described[Bianco et al., 1999;Paine et al., 2000].

EXAMPLE 6 In Vivo Anti-Tumor Effect of the Immune Response Induced byCripto Vaccines in Animal Models

The prophylactic or therapeutic potential of vaccines containing thehuman Cripto protein, Cripto peptides, or Cripto gene can be evaluatedin mice challenged with syngeneic murine tumor cell line that expressCripto. The tumor cell lines could be a murine tumor cell linetransfected with the human Cripto gene. For instance the transfectedcell lines could be the TC1 cell line transfected with human Criptogene. On the other hand, the high level of homology between human Criptoand its murine homologue suggests that cross-reactive immune responsesinduced by the human vaccine can protect against mouse Cripto-expressingtumors. Indeed, Cripto gene (TDGF-1) encodes a 171-amino acid proteinwhich has 93% identity with its human counterpart [Liguori et al.,1996]. Therefore mice could be protected by immunization with a humanCritpo vaccine from murine tumor challenge or spontaneously arisingtumor known to endogenously over-express the Cripto protein. With thisrespect, spontaneous tumors in transgenic mice designed to over-expressseveral different oncogenes such as MMTV-Polyoma virus middle T antigen,MMTV-c-ErbB2, and MT-hTGF alpha, have been shown also to over-expressCripto [Kenney et al., 1996;Niemeyer et al., 1999].

Alternatively, in order to vaccinate with the syngeneic gene, thetumor-bearing mice could be vaccinated with a plasmid DNA or a viraldelivery system, e.g. adenoviral vector, encoding the murine Criptoprotein to protect from murine tumor growth.

6.1: Prophylactic Experimental Design:

Mice would be vaccinated on day 0 and 14, prior tumor challenge, byeither intra-footpad injections of 5 μg of Cripto protein in adjuvant,intra-dermal injections of DNA plasmid encoding Cripto using gene-guntechnology, or intra-dermal injections of a viral vector, e.g.adenoviral vector, encoding Cripto. Then, 10⁶ TC1-CR-1 cells, humanCripto-expressing tumor cells, could be injected subcutaneously in theflank of C57BL/6 immunocompetent mice 1 or 2 weeks post vaccination. Thetumor growth should be monitored in vivo by measuring individual tumorstwice a week for several weeks post-tumor challenge.

On the other hand, the efficacy of prophylactic vaccination could beassessed by inhibiting the development of spontaneous Cripto-expressingtumors in transgenic mice [Niemeyer et al., 1999] immunized multipletimes before onset of the palpable tumor with either form of Criptovaccines.

6.2: Therapeutic Experimental Design:

10⁶ TC1-CR-1 cells, human-Critpo expressing tumor cells, would beinjected subcutaneously in the flank of C57BL/6 immunocompetent mice.Mice could be vaccinated on days 7 and 14, post-tumor challenge, byeither intra-footpad injections of 5 μg Cripto protein in adjuvant,intra-dermal injections of DNA plasmid encoding Cripto using gene-guntechnology, or intra-dermal injections of a viral vector, e.g.adenoviral vector, encoding Cripto. The tumor growth should be monitoredin vivo by measuring individual tumors twice a week. One to 4 weeksafter the second immunization, several mice per group will be sacrificedto harvest spleen cells, draining lymph nodes, and sera for analysis ofthe immune responses to establish a correlation between the induction ofa Cripto-specific immune response and the anti-tumor effect. Theanalysis of the Cripto-specific immune response induced by immunizationcould be assessed by measuring the antibody titers, antibody isotypicprofile, the CD4⁺ T-cell proliferation response, and CTL CD8⁺ T-cellresponses including cytokines production and lysis activity againstCripto-expressing target cells. All assays would be performed accordingto standards protocols.

A similar kind of experiment could be carried out by challengingparental mice with transplantable murine mammary tumor linesover-expressing Cripto. These cells would be established fromspontaneous tumor arising in various oncogene transgenic mouse strainssuch as MMTV-Polyoma virus middle T antigen, MMTV-c-ErbB2, and MT-hTGFalpha transgenic mice [Niemeyer et al., 1999].

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1. An immunogenic composition comprising: (a) a component selected fromthe group consisting of: physiologically acceptable carriers,immunostimulants, and adjuvants; and (b) a polypeptide selected from thegroup consisting of: (i) a polypeptide consisting of the amino acidsequence of SEQ ID NO:11; and (ii) a polypeptide consisting of the aminoacid sequence of SEQ ID NO:12.
 2. The immunogenic composition accordingto claim 1 comprising a TH-1 inducing adjuvant.
 3. The immunogeniccomposition according to claim 2 wherein the TH-1 inducing adjuvant isselected from 3D-MPL, QS21, a mixture of QS21 and cholesterol, a CpGoligonucleotide, and a mixture of two or more of said adjuvants.